Treatment and prevention of ocular neurodegenerative disorder

ABSTRACT

The invention relates to the use of a pharmaceutical composition containing nicotinamide (NAM) and/or pyruvate as a neuroprotective medicament or gene therapy in the treatment of neurodegenerative disorders, in particular axon degeneration of neuronal tissue in ocular-related neurodegeneration diseases including glaucoma.

REFERENCE TO RELATED APPLICATION

This is a continuation application of the International PatentApplication No. PCT/US2016/058388, filed on Oct. 24, 2016 and publishedas WO2017/070647, which claims the benefit of the filing date under 35U.S.C. § 119(e) to U.S. Provisional Application Nos. 62/245,467, filedon Oct. 23, 2015, and 62/366,211, filed on Jul. 25, 2016, the entirecontents of each of which are hereby incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The present invention was made with U.S. government support under GrantNo. R01 EY011721, awarded by the National Institute of Health (NIH). TheU.S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Axon injury is an early event in neurodegenerative diseases.Neurodegenerative diseases are characterized by a dysfunction or loss ofviable nerve cells from either the peripheral or the central nervoussystem. In many cases axon degeneration is shown to precede neural loss,which is a process that is invariably more pronounced at the distalrather than the proximal end of axonal processes. Upstream molecularsignals that trigger the neurodegeneration cascades in the neuron remainunknown.

There are reports suggesting the use of nicotinamide adeninedinucleotide (NAD) and its related compounds to reduce axondegeneration. For example, US 2006-0211744 A1 (the '744 application)describes the use of agents, including NAD, NADH, and nicotinamide(NAM), to reduce chronic neural degeneration. Using a cell culture modelof transected dorsal root ganglion (DRG) neuron axon, the '744application discloses that NAD provided a protecting effect to theseneuron against axon degeneration. The '744 application further disclosesthat NAM reduces neural degeneration in mouse model for ExperimentalAutoimmune Encephalomyelitis (EAE), Amyotrophic Lateral Sclerosis (ALS),and Relapsing Remitting Multiple Sclerosis (RRMS). U.S. Pat. No.7,776,326 (the '326 patent) discloses a method of treating axonaldegradation in neuropathic diseases in mammals by administering agentsthat increases NAD activity in the injured neurons. Using primary cellcultures of dorsal root ganglion (from spinal nerves) and axotomy(mechanical cut) at a location dose to soma, the '326 patenteesexplicitly concluded that nicotinic acid and NAM did not work todiminish axonal degeneration. In an eye injection study, the '326patentees again stated that, when injected intravitreally, NAM did notshow any difference from the control animals.

Glaucoma is one of the most common neurodegenerative diseases, andrepresents the leading cause of irreversible blindness, affecting over70 million people worldwide (Quigley and Broman, Br J Ophthalmol 90,262-267, 2006), especially in the elderly. Glaucoma is a complex,multifactorial disease characterized by the progressive dysfunction andloss of retinal ganglion cells (RGCs) leading to vision loss. Highintraocular pressure (IOP) and increasing age represent susceptibilityfactors for neurodegeneration for glaucoma. Recent advances have hintedto a variety of molecular changes occurring in glaucomatous tissues atthe early stages of disease (Howell et al., Journal of ClinicalInvestigation 121, 1429-1444, 2011; Nickells et al., Annu Rev Neurosvi35, 153179, 2012). Early cellular and molecular mechanisms that initiateglaucomatous damage within RGCs are poorly known. It appears that theRGCs are insulted at multiple sites very early in glaucoma includingchanges affecting their cell bodies, dendrites and synapses in theretina'as well as their axons in the optic nerve. The mechanism how highIOP and aging drive neuronal vulnerability and initiate glaucoma inhumans is still unclear.

Although there are available strategies to alleviate elevated IOP, thereare no effective treatments or preventive measures targeting ocularneural degeneration. Accordingly, there is a continuing need indeciphering the upstream molecular signals that trigger the initialneurodegenerative process as well as identifying new molecular targets,so as to provide therapeutic interventions for reducing RGC damage inthe retina and axon degeneration, particularly in the treatment ofglaucoma.

SUMMARY OF THE INVENTION

The present invention is primarily predicated on our finding that thereis potential effect of nicotinamide (NAM) or pyruvate, or a combinationthereof, to protect neuronal cell body, axon, and an associated celltype, thus being beneficial to the treatment of neurodegeneration (e.g.,axon degeneration) and ocular neurodegeneration diseases (e.g.,glaucoma).

It is an object of the present invention to provide a method of treatingor preventing glaucoma, comprises the step of administering to a subjectin need of treatment of a pharmaceutical composition comprising atherapeutic effective amount of NAM and/or pyruvate.

It is an object of the present invention to provide a method of loweringintraocular pressure, e.g., in glaucoma, comprises the step ofadministering to a subject in need of treatment of a pharmaceuticalcomposition comprising a therapeutic effective amount of NAM and/orpyruvate.

It is an aspect of the present invention to provide a method ofimproving visual function in patients having or suffering from glaucoma,comprises the step of administering to a subject in need of treatment ofa pharmaceutical composition comprising a therapeutic effective amountof NAM and/or pyruvate.

The present therapy treatment (medicaments and/or gene therapy), withoutbeing hound by a particular theory, is believed to work by lowering theintraocular pressure by two main mechanisms: 1) reducing aqueous humorproduction, and/or 2) increasing aqueous humor outflow. The presenttherapy can serve as an effective treatment by lowering the high IOP,thus preserving the axon such as optic nerve, and preventing thesubsequent loss of visual function.

The present therapy treatment is useful to treat or preventneurodegeneration in glaucoma.

The present glaucoma treatment is effective in treating or preventingneural dysfunction and neurodegeneration at the level of retina (e.g.,RGCs). Glaucoma can exist at any levels of intraocular pressure (highIOP or normal IOP). The present therapy treatment provides anIOP-independent effect of neuroprotective effect on retinal cells suchas the retinal ganglionic cells (RGCs).

In certain embodiments, the present treatment prevents and/or delays theprogression of glaucoma (e.g., primary open angle glaucoma or POAG) viareducing the intraocular pressure. Almost all current strategies fortreating glaucoma are aimed at lowering or preventing a rise in IOP.

In certain embodiments, the present treatment prevents and/or delays theprogression of glaucoma (e.g., primary open angle glaucoma or POAG) viaboth providing direct neuroprotection and reducing the intraocularpressure. The present therapy provides an unexpected dual benefit in thetreatment of glaucoma.

In certain embodiments, the present treatment prevents and/or delays theprogression of glaucoma without reducing intraocular pressure. Thus thepresent therapy method provides an unexpected advantage, in that themedicament and/or gene therapy offers an IOP-independent neuroprotectiveeffect.

The present therapy method provides a treatment for glaucoma, such asprimary open angle glaucoma, by protecting optic nerve changes, andcharacteristic patterns of visual field loss. According to the PreferredPractice Patterns of AAO, two of the three findings (elevated IOP, opticnerve damage, or visual field loss) must be present for the diagnosis ofprimary open angle glaucoma.

It is an object of the invention to provide a method of treating aneurodegenerative disorder (such as an ocular neurodegenerative disease,e.g., glaucoma) in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising one or more compounds thatincrease intracellular level of NADt, or that increases intracellularlevel of NAD⁺, NADH, GSH, GSSG, PQQ, or pyruvate.

In certain embodiments, the neurodegenerative disorder (such as theocular neurodegenerative disease, e.g., glaucoma) involves axondegeneration, neuronal dysfunction, somal shrinkage, synapse loss,dendritic atrophy, and/or normal neuronal aging. In certain embodiments,the neurodegenerative disorder (such as the ocular neurodegenerativedisease, e.g., glaucoma) involves axon degeneration.

In certain embodiments, the neurodegenerative disorder (such as theocular neurodegenerative disease, e.g., glaucoma) involves Walleriandegeneration, Wallerian-like degeneration or dying back axondegeneration. For example, the Wallerian degeneration results fromneuronal injury. The neuronal injury may result from disease, trauma, achemotherapeutic agent, or neuronal aging.

In certain embodiments, the neurodegenerative disorder is one or more ofAlzheimer's disease, multiple sclerosis, diabetic neuropathy, traumaticbrain injury, ischemia, peripheral neuropathy, or an ophthalmic disordersuch as glaucoma or an age-related ocular disease (e.g., age-relatedmacular degeneration (AMD), Leber's optic neuropathy, dominant opticatrophy, cataract, diabetic eye disease/diabetic retinopathy, retinaldegeneration, dry eye, low vision).

In certain embodiments, the compounds of the present invention comprisea nicotinamide adenine dinucleotide (NAD⁺) precursor (e.g., nicotinicacid (Na), nicotinamide (NAM), nicotinamide mononucleotide (NMN),nicotinamide riboside (NR), or a combination thereof).

In certain embodiments, the compounds of the present invention includenicotinamide, pyruvate, or pyrroloquinoline quinone (PQQ). The compoundsof the present invention are believed to replenish intracellular NADt orimprove the mitochondrial electron transport chain.

In certain embodiments, the compounds of the present invention include:(a) NAM; (b) pyruvate; (c) PQQ; (d) NAM and pyruvate; (d) NAM and PQQ.

It is an object of the invention to provide a method of treating aneurodegenerative disorder (such as the ocular neurodegenerativedisease, e.g., glaucoma) in a subject in need thereof, the methodcomprising administering to the subject a gene composition. The genecomposition contains a gene that increases the expression of (e.g.,Nmnat-1, Nmnat-2, or Nmnat-3).

In certain embodiments, the gene is NMNAT1.

In certain embodiments, the gene is Wld^(S).

In certain embodiments, the method comprises administering apolynucleotide encoding Nmnat (e.g., Nmnat-1 Nmnat-2, or Nmnat-3) and/orWld^(S) to the eye of a subject.

In certain embodiments, the polynucleotide is administered to thesubject locally.

In certain embodiments, the polynucleotide is administered to thesubject on a viral vector (e.g., an AAV vector, an adenoviral vector, alentiviral vector, a retroviral vector, etc.).

In certain embodiments, the neurodegenerative disorder is glaucoma or anage-related ocular disease; the subject is a human.

It is an object of the invention to provide a method of treating aneurodegenerative disorder in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of one or more compounds that increase intracellular level ofNADt, and further comprises administering a polynucleotide encoding andexpressing Nampt, Nmnat (e.g., Nmnat-1), and/or Wld^(S) to the subject.

It is an objective of the invention to provide a method of treatingglaucoma in a subject in need thereof, comprising the step ofadministering to the subject a pharmaceutical composition containing atherapeutically effective amount of nicotinamide (NAM), thereby treatingglaucoma.

It is a further related but distinctive objective of the invention toprovide a method of preventing glaucoma in a subject in need thereof,comprising the step of administering to the subject a pharmaceuticalcomposition containing a therapeutically effective amount ofnicotinamide (NAM), thereby preventing glaucoma.

In certain embodiments, said NAM is present in a therapeuticallyeffective amount to reduce neurodegeneration in a retinal ganglion cell.For example, in certain embodiments, said pharmaceutical compositioncontains about 0.5-10 grams NAM, about 1-5 grams NAM, or about 2.5 gramsNAM for daily consumption.

In certain embodiments, said NAM is present in a therapeuticallyeffective amount to reduce intraocular pressure. For example, in certainembodiments, said pharmaceutical composition contains about 2-25 gramsNAM, about 10-20 grams NAM, or about 10 grams NAM for daily consumption.

In certain embodiments, said pharmaceutical composition furthercomprises pyruvate. Preferably, said NAM and pyruvate are present intherapeutically effective amounts to reduce neurodegeneration in aretinal ganglion cell. For example, in certain embodiments, saidpharmaceutical composition independently contains (1) about 0.5-10 gramsNAM, about 1-5 grams NAM, or about 2.5 grams NAM for daily consumption;and (2) about 0.5-10 grams pyruvate, about 1-5 grams pyruvate, or about2.5 grams pyruvate for daily consumption. Preferably, said NAM andpyruvate are present in therapeutically effective amounts to reduceintraocular pressure. For example, in certain embodiments, saidpharmaceutical composition independently contains (1) about 2-25 gramsNAM, about 10-20 grams NAM, or about 10 grams NAM for daily consumption;and (2) about 2-25 grams pyruvate, about 10-20 grams pyruvate, or about10 grams pyruvate for daily consumption.

In certain embodiments, said pharmaceutical composition furthercomprises one or more compounds selected from the group consisting of:nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ),nicotinamide adenine dinucleotide (NAD), and nicotinamide ribose (NR).In certain embodiments, when PQQ is present, the pharmaceuticalcomposition comprises about ˜10 mg-10 g, about 50 mg-1 g, or about 500mg PQQ for daily consumption.

In certain embodiments, the present method (with or without pyruvate)further comprises the step of administering a gene composition, whereinsaid gene composition comprises a polynucleotide encoding NMNAT1. Incertain embodiments, the polynucleotide is in a viral vector, such as anadeno-associated virus (AAV) vector, an adenoviral vector, a lentiviralvector, or a retroviral vector.

In certain embodiments, said viral vector is AAV. In certainembodiments, said viral vector is AAV2.2. In certain embodiments, saidviral vector is a lentiviral vector.

In certain embodiments, said gene composition is administeredintravitreally intraocularly. Preferably, said gene composition isadministered intravitreally.

In certain embodiments, said subject is a human subject.

In certain embodiments, said subject has an intraocular pressure ofabout 12-21 mmHg. In certain embodiments, said subject has anintraocular pressure of greater than 21 mmHg.

In certain embodiments, said subject has not developed neurodegenerationsymptoms of glaucoma. In certain embodiments, said subject has developedneurodegeneration symptoms of glaucoma.

In certain embodiments, said subject has developed visual dysfunction.

In certain embodiments, the method further comprises administering tothe subject an additional therapeutic agent. An exemplary additionaltherapeutic agent includes an intraocular pressure lowering agent. Incertain embodiments, said additional therapeutic agent is a betablocker, a nonselective adrenergic agonist, a selective α-2 adrenergicagonist, a carbonic anhydrase inhibitor, a prostaglandin analog, apara-sympathomimetic agonist, a carbachol or a combination thereof. Incertain embodiments, said additional therapeutic agent is timolol,levobunolol, metipranolol carteolol, betaxolol, epinepherine,apraclonidine, brimonidine, acetazolamide, methazolamide, dorzolamide,brinzolamide, latanoprost, travaprost, bimataprost, pilocarpine,echothiophate iodine, carbachol, or a combination thereof.

It is yet an objective of the invention to provide a method of improvingvisual function in a subject in need thereof, comprising the step ofadministering to the subject a pharmaceutical composition containing atherapeutically effective amount of nicotinamide (NAM), therebyimproving visual function.

In certain embodiments, said pharmaceutical composition furthercomprises pyruvate.

In certain embodiments, the method further comprises the step ofadministering a gene composition, wherein said gene compositioncomprises a polynucleotide encoding NMNAT1.

It should be understood that all embodiments described herein can hecombined with any other embodiment unless explicitly disclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of Hierarchical Clustering (HC), for definingmolecularly determined stages of glaucoma in DBA/2J (D2) mice at initialstages of disease and morphologically indistinguishable from age-matchedD2-Gpnmb⁺ or young controls, HC was based on RNA-sequencing in theretinal ganglion cells (RGCs) isolated from the D2 mice and thecontrols. HC allows the clustering of the RGC samples into distinctgroups with control and young samples being molecularly similar(Spearman's rho). Circles=samples from D2 RGCs, triangles=samples fromD2-Gpnmb⁺ RGCS. Inset: number of differentially expressed (DE) genes(q<0.05) between D2-Gpnmb⁺ and each group.

FIG. 2A shows the number of differentially expressed (DE) genes amongthe 4 D2 mice groups (Group 1, Group 2, Group 3, and Group 4) and theD2-Gpnmb⁺ control group.

FIG. 2B shows heatmap correlations of all samples (Spearman's rho,blue=highest correlation, red=lowest correlation). Dendrogram from FIG.1 is shown in grey.

FIG. 3A shows that the mitochondrial:nuclear read total ratio increaseswith increasing HC distance from controls (9-month old mice). The resultis consistent with the notion that mitochondrial dysfunction is an earlydriver of RGC damage in glaucoma.

FIG. 3B shows several top significantly enriched pathways based onIngenuity Pathway Analysis (IPA), using RNA-seq data obtained from RGCsof pre-disease D2 mice (three clusters of 9-month old D2 mice—Group 2,Group 3, and Group 4) and age- and sex-matched wild-type controls(D2-Gpnmb⁺). The larger the -log p value the greater the enrichment. Twoof the top 3 enriched pathways are mitochondrial dysfunction andoxidative phosphorylation. See also Table 3 for additional significantlyenriched pathways. Note that there are no differentially expressedpathways in D2 Group 1.

FIG. 3C shows that transcript expression primarily increases for nuclearencoded mitochondrial proteins with increasing HC distance of the D2groups from controls (all 9-month old mice). The result is againconsistent with the notion that mitochondrial dysfunction is an earlydriver of RGC damage in glaucoma. Dots represent individual genes, greyor lighter dots=not differentially expressed, red or darkerdots=differentially expressed at q<0.05, Genes were taken from mouseMitoCarta2.0 (Calvo et al., Nucleic Acids Res 44, D1251-1257, 2016).

FIG. 3D shows that RNA-sequencing identifies increased mitochondrialfission gene transcripts early in glaucoma (9-month old mice). Theresult is consistent with the notion that mitochondrial dysfunction isan early driver of RGC damage in glaucoma.

FIG. 3E shows an early mitochondrial unfolded protein response comparedto controls. Data shown is for D2 Group 4 (9-month old mice). The resultis consistent with the notion that mitochondrial dysfunction is an earlydriver of RGC damage in glaucoma.

FIG. 3F represents results of individual gene expression plots showingmetabolic and oxidative events in early glaucoma (9-month old mice).Dots represent individual genes, grey or lighter dots=not differentiallyexpressed, red or darker dots=differentially expressed at q<0.05. Again,oxidative phosphorylation genes are among the ones showing the highestlevels of differential expression, and differential expression appearsto be progressively higher in D2 groups having increasing HC distancefrom the control (Group 4>Groups 3 and 2).

FIGS. 3G and 3H are bar graphs summarizing data from Western blotprotein validation (FIGS. 3I and 3J, respectively) of differentiallyexpressed genes (n=4/age group). There were general protein increases inboth cytochrome c (FIG. 3G) and eIF2α (FIG. 3H) that correlated withtranscript abundances as assessed by RNA-sequencing. The vertical axisin both bar graphs is relative density to β-actin. *=P<0.05, **=P<0.01.

FIG. 3K shows that oxidative phosphorylation genes are differentiallyexpressed across all groups, with increasingly higher expressions in theD2 groups having increasing HC distance from the control (Group 4>Group3>Group 2>Group 1), Red=highest expression, blue=lowest expression,I-V=mitochondrial complexes I-V (tabulated in Table 1), G=D2-Gpnmb⁺, 1-4D2 Groups 1-4.

FIGS. 4A and 4B show that 9-month old D2 mice have decreased cristaevolume in RGC somal and dendritic mitochondria. However, there is nosignificant difference in total mitochondrial size/volume. Scale bar=350nm. **=P<0.01, *=P<0.001.

FIG. 5A shows that NAD(t) levels are increased in NAM^(Lo)-treated (550mg/kg/day) D2 mice (n=22/group). *=P<0.05, ***=P<0.001.

FIG. 5B shows that GSH/GSSG levels decrease with age (n=22/group).*P<0.05, ***=P<0.001.

FIG. 5C shows that NAD(t) levels also decrease with age in D2-Gpnmb⁺control mice (n=22/group). ***=P<0.001.

FIG. 5D shows a decrease in pyruvate levels with age that is restored bypyruvate treatment. D2 mice were treated with 500 mg/kg/d pyruvate innormal drinking water from 6-month of age.

FIG. 5E shows a decrease in total NAD (NAD(t)) (i.e., NAD⁺+NADH) in D2retinas with age that is restored by NAM treatment (550 mg/kg/day) orthe addition of the Wld^(S) allele. Combination of Wld^(S) and NAMaffords additional benefit.

FIGS. 6A and 6B show that NAM treatment (550 mg/kg/day) lessensupregulation in early glaucoma as assessed by immunostaining(n=6/group). *=P<0.05, ***=P<0.001. The result demonstrates that NAMtreatment prevents early damaging changes in D2 glaucoma.

FIG. 7A shows that RNA-sequencing identifies changes to cellularmetabolism—specifically, fatty acid metabolism gene changes in D2glaucoma. Dots represent individual genes, grey or lighter dots=notdifferentially expressed, red or darker dots=differentially expressed atq<0.05.

FIG. 7B shows inner-retina extracellular lipid droplet formation inIOP-insulted, aged D2 eyes as stained using Oil Red O. Staining waspresent in D2 eyes which had both no (NOE) or severe (SEV) optic nervedegeneration. Extra-ocular fat was used as a positive control(n=6/group), Scale bar=25 μm.

FIGS. 8A and 8B show increased levels of DNA damage as assessed by γH2AXstaining (n=6/group). The result is represented as bar graph of γH2AXstaining max intensity (AU). ***=P<0.001.

FIGS. 9A and 9B show that NAM prevents PARP activation in glaucoma. PARPis a major constumer of NAD⁺, and may contribute to NAD(t) decreases inRGCs with age. After administration of NAM (550 mg/kg/day), PARPexpression (pan-PARP immunostaining) is reduced in NAM-treated retinas(n=6/group). ***=P<0.001. Scale bar 15 μm.

FIGS. 10A & 10C (IOP profiles), and FIGS. 10B & 10D (clinicalpresentation of IOP elevating iris disease) show that protectivestrategies do not change clinical disease progression/presentation intreated eyes. Iris disease progressed at a similar rate and reached asevere state in all groups within the same timeframe. NAM^(Lo)=550mg/kg/day. NAM^(Hi)=2000 mg/kg/day. Nmnat1=gene therapy by expressingexogenous Nmnat1 (on viral vector).

FIG. 11A shows that NAM protects from optic nerve degeneration. Green orlower sections of the bars=no or early, glaucoma (NOE) (a stage with nonerve damage), yellow or middle sections of the bars=moderate damage(MOD), red or top sections of the bars=severe (SEV) damage. Fisher'sexact test: **=P<0.01, ***P<0.001. NAM^(Lo)=550 mg/kg/day. NAM^(Hi)=2000mg/kg/day. Early start=treatment starts at 6 months of age (pre-elevatedIOP in almost all eyes). Late start treatment starts at 9 months of age(following onset of elevated IOP; at this time-point the majority ofeyes have or have had elevated IOP).

FIG. 11B shows that nicotinamide protects against optic nervedegeneration in D2 glaucoma at 12 months of age. Chart shows percentageof nerves with no detectable glaucoma (NOS; lower sections of the bars),moderate glaucomatous damage (MOD; middle sections of the bars), orsevere glaucomatous damage (SEV; top sections of the bars). From left,DBA/2J control—D2 mice on standard drinking water; Nicotinamide(NAM^(Lo)) early start—D2 mice on standard drinking water supplementedwith 550 mg/kg/day NAM from 6 months of age (pre-disease); Nicotinamide(NAM^(Lo)) late start—D2 mice on standard drinking water supplementedwith 550 mg/kg/day NAM from 9 months of age (during disease);Nicotinamide (NAM^(Hi)) early start—D2 mice on standard drinking watersupplemented with 2,000 mg/kg/day NAM from 6 months of age(pre-disease); Pyruvate—D2 mice on standard drinking water supplementedwith 500 mg/kg/day pyruvate from 6 months of age (pre-elevated IOP);NAM^(Lo)+Pyruvate—D2 mice on standard drinking water supplemented with550 mg/kg/day NAM+500 mg/kg/day pyruvate from 6 months of age(pre-disease); D2 Wld^(s)—D2 mice carrying the Wld^(S) transgene(altered NMNAT enzyme that enhances enzymatic activity) on standarddrinking water; D2. Wld^(S) +Nicotinamide (NAM^(Lo))—D2 mice carryingthe Wld^(S) transgene (altered NMNAT enzyme) on standard drinking waterwith 550 mg/kg/day NAM from 6 months of age (pre-elevated IOP) Note thatin addition to treating glaucomatous neurodegeneration interventionally,NAM also protects against glaucomatous neurodegenerationprophylactically.

FIG. 12A shows that NAM and pyruvate protects from RGC soma loss(n=8/group), retinal NFL and IPL thinning (n=9/group), optic nervedegeneration (n>50/group), and loss of anterograde axoplasmic transport(n=20 /group). Corresponding markers and color keys are beneath eachcolumn. Scale bars: RBPMS=20 μm, Nissl=20 μm, PPD=20 μm, CT-β=100 μm(retina), 200 μm (LGN, Sup, Col.), ONH=optic nerve head, LGN=lateralgeniculate nucleus, Sup. Col.=superior colliculus. White asteriskdenotes loss of axonal transport at the site of the ONH.

FIG. 12B shows recovery of axonal transport in NAM and pyruvate treatedD2 mice. Loss of axonal transport is a prominent feature in glaucoma andcan be used as a metric of neuronal health. Using a fluorescentlylabeled cholera toxin (Ct-B; green) axonal transport from the eyeportion of the retinal ganglion cell to the terminal (brain) end of thecell can be visualized. Top row: D2.Gpnmb^((wt)) mice have normal,complete axonal transport from the retina to visual centers in the brain(LGN; lateral geniculate nucleus, Sup. col.; superior colliculus).Second row: D2 mice have incomplete axonal transport halting within theoptic nerve (white asterisk). There is no labeling present in the visualcenters of the brain. Third and forth rows: NAM treatment (third row) orpyruvate treatment (forth row) prevent axonal transport loss.

FIG. 13A shows that NAM protects from RGC soma loss (n=8/group, thedensity drop between D2 and D2-Gpnmb⁺ is due to pressure inducedstretching). ***=P<0.001.

FIG. 13B shows NAM^(Lo) (550 mg/kg/day) and pyruvate (500 mg/kg/day)prevent RGC soma loss (using a specific marker of RGCs; RBMPS).

FIG. 14 shows that NAM (NAM^(Lo) and NAM^(Hi)), pyruvate (500mg/kg/day), Wld^(S), and combinations thereof all protect D2 mice fromearly visual function loss based on visual function testing using PERG(pattern electroretinography) amplitude (n>20/group). PERG is a verysensitive and early measure of glaucoma, thus NAM or pyruvate treatmentprevents even the very early stages of glaucoma. The combination ofNAM^(Lo)+pyruvate, and the combination of NAM^(Lo)+Wld^(S), both showadditional recovery of PERG amplitude, NAM^(Lo)=550 mg/kg/day.NAM^(Hi)=2,000 mg/kg/day.

FIG. 15 shows that NAM^(Lo) (550 mg/kg/day) and pyruvate (500 mg/kg/day)protect PERG even in aged mice. Example traces from 6-month and 12-monthD2 and 12-month NAM^(Lo)-treated mice (550 mg/kg/day) are shown. Resultsfor 12-month pyruvate-treated mice, and NAM^(Lo)-treated Wld^(S) miceare also shown.

FIGS. 16A and 16B show that NAM^(Lo)-treatment (550 mg/kg/day) preventssynapse loss in early glaucoma as assessed by SNAP-25 staining(n=6/group). **=P<0.01, ***=P<0.001. Scale bar=25 μm.

FIGS. 17A and 17B show that NAM (NAM^(Lo)=550 mg/kg/day) prevents earlymitochondrial dysfunction in dendritic mitochondria as shown in FIGS. 4Aand 4B. These data correspond with early changes to PERG and previouslyreported synapses loss in D2 retinas at 9-month. Thus, elevated IOPinduced mitochondrial dysfunction may drive early neurodegenerativechanges. **=P<0.01, ***P<0,001. ns=not statistically significant. Barscale=350 nm.

FIG. 18 shows that NAM treatment (550 mg/kg/day) prevents lipid dropletformation in the inner retina (12-month; as in FIG. 7B). Scale bars=25μm.

FIGS. 19A and 19B show that NAM treatment (NAM^(Lo)=550 mg/kg/day)prevents DNA damage in early glaucoma as assessed by γH2AX staining(n=6/group). ***=P<0.001. Scale bar=25 μm.

FIG. 20A represents individual gene expression plots showing metabolicand DNA damage pathways are returned to normal in NAM-treated RGCS. Dotsrepresent individual genes, grey or lighter dots=not differentiallyexpressed, red or darker dots=differentially expressed at q<0.05compared to D2-Gpnmb⁺ control.

FIG. 20B is a heatmap of gene expression (all expressed genes) showingthat NAM-treated RCGs are molecularly similar to controls.

FIG. 21A shows NAM-treated RCGs are molecularly similar to both youngand age-matched no glaucoma control RCGs. (Spearman's rho).Circles=samples from D2 RGCs, triangles=samples from D2-Gpnmb⁺ RGCs,squares=samples from NAM-treated (NAM^(Lo)=550 mg/kg/day) RGCs. Thus NAMtreatment prevents disease and age-related molecular changes.

FIG. 21B summarizes the number of differentially expressed genes amongthe D2 groups (Group 1, Group 2, Group 3, and Group 4) compared to thecontrol D2-Gpnmb⁺ group.

FIGS. 21C and 21F show that NAM-treatment (NAM^(Lo)=550 mg/kg/day)prevents transcriptome imbalances and OXPHOS imbalances(mitochondial:nuclear library size ratio) seen in 9-month 132 RGCs. InFIG. 21F, Red=highest expression, blue=lowest expression.I-V=mitochondrial complexes I-V (tabulated in Table 1), G=D2-Gpnmb⁺,1-4=D2 Groups 1-4, N=NAM.

FIG. 21D D2 mice were treated with 550 mg/kg/day nicotinamide in normaldrinking water from 6-month of age (Aged NAM). RNA-seq of retinalganglion cells from NAM-treated mice shows that NAM prevents age- anddisease-related gene expression changes. NAM-treated retinal ganglioncells are most similar to young (Young Control) rather than age-matchedcontrols (Aged Control). This indicates that NAM prevents, theage-dependent molecular changes.

FIG. 21E shows heatmap correlations of all samples (Spearman's rho,blue=highest correlation, red=lowest correlation). Dendrogram from FIG.21A is shown in grey.

FIGS. 22A and 22B show that NAM is protective against neurodegenerativetreatments that model glaucomatous insults. NAM showed an attractivedose-response effect in protecting ganglion cell layer cells (GCL cells)from death (FIG. 22A) and pre-apoptotic nuclear shrinkage (FIG. 22B). Inan axotomy culture model of retinal ganglion cell damage, addition ofnicotinamide (NAM) protects against cell shrinkage (sign of cellapoptosis and dysfunction) in a dose-dependent manner (5 dayspost-axotomy). Mean nuclear diameter of DAPI-labeled nuclei from D2 miceretinas exposed to different concentrations of NAM for 5 days wasmeasured (including Day 0 control (normal retina), No treatment (5 dayspost-axotomy), 100 mM and 500 mM NAM (5 days post-axotomy). NAM treatedretinas were indistinguishable from uninjured, baseline controls (Day0). There were also significant protective effects treating retinas withβ-NAD & β-NMN (n=8 retinas/group). ***=P<0.001. Scale bar=20 μm.

FIGS. 23A-23C show that NAM (NAM^(Lo)=550 mg/kg/day) prevents PERG (FIG.23A) and soma loss (FIGS. 23B and 23C) in TNFα injected eyes at 12-weekpost-TNFα administration (n=20/group). *=P<0.05. ns=not statisticallysignificant, Scale bar=20 μm.

FIGS. 24A and 24B show that gene therapy robustly protects fromglaucomatous neurodegeneration. D2 eyes were intravitreally injected at5.5-month with AAV2.2 viral vector carrying a plasm id to overexpressmurine Nmnat1 under a CMV promoter. FIG. 24A shows that Nmnat1overexpression prevents RGC soma loss and loss of anterograde axoplasmictransport (n=10/group), as demonstrated in FIG. 12A. Scale bar is 50 μmin FIG. 24A, and is 100 μm in FIG. 24B.

FIGS. 24C and 24D show that Nmnat1 gene therapy also protects D2 eyeswith elevated IOP against optic nerve degeneration (n>40/group) (FIG.24C), soma loss (n=8/group) (FIG. 24D, top panel), and PERG amplitude(n>20/group) (FIG. 24D, bottom panel). Addition of NAM (NAM^(Lo)=550mg/kg/day) in drinking water afforded additional protection againstoptic nerve degeneration (Nmnat1 compared to Nmnat1+NAM^(Lo)=P<0.001,Fisher's exact test) (FIG. 24C). **P<0.01, ***=P<0.001.

FIG. 25 shows the NAD recycling pathway with NAM and NMNAT1/WLD^(S).

FIGS. 26A and 26B show that PQQ administration prevents nuclear diametershrinkage (FIG. 26A), and a decrease in cell density (FIG. 26B), inaxotomized retinas treated with PQQ.

FIGS. 27A-27E show FACS sorting RGCs. Retinal samples were stained withan antibody cocktail (see Materials and Methods). FIG. 27A shows forwardand sideward scatter (see FIG. 27A insert and FIG. 27B) that are gatedto identify live cells (FIG. 27B insert). RGCs were identified as gatedThy1.2⁺ (Cd11b⁻, Cd11c⁻, Cd31⁻, Cd34⁻, Cd45.2⁻, GFAP⁻, DAPI⁻) events.Only Cd11b, Cd45, and Thy1.2 plots are shown (FIGS. 27C and 27D. In FIG.27E, FACS positive RCGs were plated and stained with SNAP-25 andβ-tubulin to confirm RGC status. Scale bar=25 μm.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this application shall have the following meanings.

As used herein, the term “glaucoma” refers to an eye disease thatresults in damage to the retina and optic nerve and visual dysfunctionor vision loss. Glaucoma occurs more commonly among older people. Visionloss from glaucoma is permanent and is irreversible.

As used herein the term “a subject/patient in need of treatment thereof”is generally understood to be a person that suffers from certainneurodegeneration diseases or conditions, especially from ocularneurodegeneration including glaucoma, or just feels to be in need forsuch treatment.

As used herein, the term “glaucoma” encompasses primary open angleglaucoma, secondary open angle glaucoma, normal tension glaucoma,hypersecretion glaucoma, primary angle-closure glaucoma, secondaryangle-closure glaucoma, plateau iris glaucoma, pigmentary glaucoma,combined-mechanism glaucoma, developmental glaucoma, steroid-inducedglaucoma, exfoliation glaucoma, amyloid glaucoma, neovascular glaucoma,malignant glaucoma, capsular glaucoma, plateau iris syndrome, and thelike.

As used herein, the term “normal intraocular pressure (normal “IOP”) inhumans refers to a human subject having an IOP values of 10 mmHg to 21mmHg. Some individuals, however, may develop optic nerve damage despitea normal IOP (known as normal-tension glaucoma).

As used herein, the term “high intraocular pressure” (high IOP) inhumans refers to a human subject having an IOP value greater than orequal to 21 mmHg (or 2.8 kPa). High IOP is known to be a risk factor forglaucoma. Some individuals, however, may have high IOP for years andnever develop optic nerve damage.

As used herein, the term “neuroprotective” or “neuroprotection” refersto the ability to protect neurons, their synapses, dendrites, somas, oraxons in the ocular nerve (e.g., optic nerve), central or peripheralnervous system from damage (including functional dysfunction) or death,or to delay the onset of neuronal damage or death, or alleviate theseverity of the neuronal damage extent of death among a population ofneurons.

As used herein, the term “preventing” or “prevention” with respect to,for example, neuronal damage or death in general or ocularneurodegeneration disease (e.g., glaucoma) in particular, refers to theability of the compounds or agents of the present invention to conferneuroprotection, preferably before such damage, death, or diseaseoccurs. Thus prevention of glaucoma includes avoiding the development ofglaucoma, reducing the risk or chance of eventually developing glaucoma,delaying the onset or progression of glaucoma, or reducing the severityof neuronal damage/extent of neuronal death/loss among a population ofneurons should glaucoma eventually develop.

As used herein, the term “treating” or “treatment” includes theadministration of the compounds or agents of the present invention to asubject to alleviate, arrest, or inhibit development the symptoms orconditions associated with neurodegeneration, such as glaucoma.

As used herein, “improving vision or visual function” refers to theeffect of the compounds or agents of the present invention in improvingvision or a visual function (such as a visual field test or patternelectroretinography (PERG) amplitude of RGC) in a subject administeredwith such compounds or agents, as compared to a control subject notadministered with such compounds or agents.

As used herein, the term “therapeutically effective amount” means theamount that, when administered to a subject, produces effects for whichit is administered. For example, a “therapeutically effective amount,”when administered to a subject to inhibit neurodegeneration, reduced IOPelevation, and/or improves RGC function (e.g., prevents the worsening ofRGC function).

As used herein, the term “subject” or “patient” are used interchangeablyand refer to mammals such as rodent, dog, and human. Accordingly, theterm “subject” or “patient” as used herein means any mammalian patientor subject (e.g., human) to which the compounds of the invention can beadministered.

As used herein, the term “medicament” or “pharmaceutical composition”refers to a pharmaceutical formulation that is of use in treating,curing or improving a disease or in treating, ameliorating oralleviating the symptoms of a disease.

As used herein, the term “expression vector” refers to a nucleic acidmolecule that is capable of effecting expression of a gene/nucleic acidmolecule it contains in a cell compatible with such sequences. Theexpression vectors typically include at least suitable promotersequences and optionally, transcription termination signals.

The following abbreviations are used:

-   -   NAM—nicotinamide    -   NAMPT—nicotinamide phosphoribosyl transferase    -   NMN or β-NMN—nicotinamide mononucleotide    -   NMNAT—nicotinamide mononucleotide adenylyl transferase    -   NAD—nicotinamide adenine dinucleotide    -   NaMN—nicotinic acid mononucleotide    -   NR—nicotinamide riboside

Overview

The present invention is primarily based on the use of astate-of-the-art molecular technology (RNA-sequencing; RNA-seq) onretinal ganglion cells (RGCs), which led to the discovery thatmitochondrial abnormalities are an early driver of neuronal dysfunction,occurring at a time that is prior to detectable neural degeneration.Using the DBA/2J (D2) mouse model (a chronic age-related, inheritedglaucoma), the present inventors further uncovered the relationshipbetween increasing age, a key risk factor for most glaucoma, and highIOP in driving the neuronal degeneration in RGCs.

The present inventors developed a method of delivering to retina atherapeutic level of nicotinamide adenine dinucleotide (NAD⁺) tosupplement the declining level of NAD⁺. The administration of specificpharmaceutical composition of the invention (e.g., NAM and/or pyruvate)represents a novel approach in the treatment of ocular neurodegenerationand glaucoma. The present therapeutic method may be useful in treatingother neurodegeneration including decreases where aging neurons arevulnerable to disease-related insults.

In one aspect, the present invention provides a therapeutic use oftargeted pharmaceutical compositions to treat or prevent age-dependentneurodegenerations, e.g., to treat or prevent glaucoma.

In certain embodiments, the pharmaceutical composition is effective intreating or preventing glaucoma, preferably primary open angle glaucoma,normal tension glaucoma, and primary angle-closure glaucoma. In certainembodiments, the pharmaceutical preparation of the present invention isparticularly effective for treating or preventing primary open angleglaucoma.

In certain embodiments, the method involves administering to a subjectin need of treatment/prevention of neurodegenerative disorder (e.g.,glaucoma) a therapeutic effective amount of nicotinamide (NAM).

In certain embodiments, the subject is administered with NAM incombination with pyruvate. It is believed that the NAM and/or pyruvateincrease intracellular level of NADt (total level of NAD⁺ and NADH) andthus treats or prevents the neurodegenerative disorder in the subject.

In one aspect, the present invention provides a therapeutic use of genedelivery to increase intracellular nicotinamide adenine dinucleotide toprevent neurodegeneration and thereby treat glaucoma.

In certain embodiments, the present invention provides a method of genetherapy (e.g., driving expression of nicotinamide nucleotideadenylyltransferase 1 (Nmnat1). The expression of Nmnat1 protein wasfound to be profoundly protective and acts synergistically with NAM(i.e., 84% of eyes having no glaucomatous neurodegeneration).

An advantage of the present invention is to reduce risk factor ofdedeveloping glaucoma. The present studies clearly show that NAM, aloneor in combination with other agents (such as pyruvate), reduces riskfactors for glaucoma. In control D2 mice at 12 months of age, about 60%of nerves have severe glaucoma. This represents a 0.6 risk factor ofdeveloping glaucoma. Following low dose NAM administration (550mg/kg/day in mice), this risk factor is reduced to 0.36 (˜2-fold drop inrisk factor), or to 0.06 when high dose NAM (2,000 mg/kg/day in mice) isadministered (a 10-fold decrease in risk factor).

An advantage of the present invention is the synergistic effect betweenNAM and administration of gene therapy (e.g., NMNAT1 gene therapy). Incomparison to gene therapy that alone reduces the risk factor to 0.29(˜2 fold decrease in risk factor), gene therapy in combination with NAMreduces the risk factor to 0.16 (˜4 fold decrease in risk factor). ThusNAM and gene therapy in combination synergistically reduces the riskfactor of developing glaucoma following elevated IOP.

The present finding of a protective effect for NAM (alone or incombination with other agents such as pyruvate) is unexpected. Thepresent finding is in sharp contrast to that reported in the '326 patentwhich states that NAM has no protective effects.

There are at least the following reasons that provides the basis for thedifferent findings. The first reason is that the '326 patentees used anexperimental system that bears little relevance to in vivo effect,especially in ocular-associated diseases such as glaucoma. In theirexperiments, the '326 patentees isolated the dorsal root ganglia (DRG)neurons and induced neural damage by cutting their neuritis. Thisartificial culture-induced neural injury where the mechanical cutoccurred very close to the DRG neuron cell body hardly resembles theneurodegeneration in vivo. It is recognized that initial axon damage inglaucoma occurs at a much greater distance from the soma than the DRGneuritis. The second reason is that the '326 patentee culture system didnot use retinal ganglion cells (RGCs), which are highly relevant neuronin glaucomatous neurodegeneration. The third reason is that the '326patentees used an artificial cell system instead of an intact neuraltissue (that is composed of various cell types that communicate with andsupport each other).

Lastly, the '326 patentees mentioned an in vivo complete optic nervetransection model, in which the nerve (including its surroundingsheathe) is mechanically cut. This suddenly damages the nerve andsubstantially alters the local environment in which the axons residewhich, within 14 days, causes the death of >90% of retinal ganglioncells (see www.ncbi.nlm.nih.gov/pubmed/21610673). Although the insult isin the correct place on the nerve, there are not cases of glaucoma.Overall, the approach adopted by the '326 patentees is a very artificialand rapid method of destroying retinal ganglion cells that does notrecapitulate human glaucoma. Of interest is that, in their optic nervetransection experiments, the '326 patentees intravitreally injectcompounds (including NAM) to drive the production of NAD in the eye, andthe '326 patentees explicitly concluded that NAM is not protectiveagainst axon degeneration in RGCs in this model.

All of this is of particularly relevance. Our culture experiments aremore relevant to glaucoma as we used intact retinal tissue that includedretinal ganglion cells in the their normal relationship to other retinalcell types, and the retinal ganglion cell axons were cut at the samelocation as damage occurs in glaucoma. It is believed that the damage inglaucoma occurs in true axons in the optic nerve head where the RGCaxons exit the eye and becomes the optic nerve.

In contrast, the data presented in this application clearly shows theneuroprotective effect of NAM in glaucoma. The present use of the NAM,alone or in combination with other agents (e.g., pyruvate), affords aneuroprotective effect in ocular-associated diseases such as glaucoma.In the present application, the neuroprotective effects are evidenced byat least one of the following parameters: (i) prevention of soma loss(RBPMS staining); (ii) prevention of retinal thinning and nerve fiberlayer loss (Nissl staining); (iii) prevention of optic nervedegeneration and axon loss (PPD staining); (iv) prevention of loss ofanterograde axoplasmic transport (Ct-B staining); (v) prevention of lossof visual function (PERG); (vi) prevention, of abnormal mitochondrialcristae (EM); (vii) reduction of fat droplets (Oil Red O staining); and(viii) reduction of PARP activation (PARP staining).

The present inventors further discovered that the use of NAM, alone orin combination with other agents (e.g., pyruvate), affects intracellularevents to provide a neuroprotective effects as evidenced by: (i)reduction of HIF-1α activation (HIF-1α staining); (ii) reduction ofsynapse loss (SNAP-25 staining); (iii) restoration of nuclear tomitochondrial transcript abundance (RNA-seq); and (iv) prevention ofage-related molecular/gene changes (RNA-seq).

Another advantage of the present invention is the combined use of NAMand gene therapy in delivering NAD. The gene therapy approach isbelieved to last for at least 3-5 years as evidenced by many clinicalstudies have indicated. The gene therapy affords a synergisticneuroprotective effect with NAM and/or pyruvate in glaucoma as well aslowering IOP.

With the inventions generally described above, the following sectionsprovide more detailed descriptions for further aspects of the invention.

DBA/2J Mouse Model

The present inventors chose to use of DBA/2J (D2) mouse model becausethis mouse strain develops an inherited age-related glaucoma that highlymimics human glaucoma. D2 mice are one of the most studied models ofglaucoma with many established similarities to human glaucoma, includinginduction of the same disease mediating molecules (for examplecomplement component molecules), the same location of a key glaucomainsult in the optic nerve head, and the same topographical pattern ofRGC death as occurs in human glaucoma. The D2 mice have iris disease andhigh intraocular pressure starting at about 6-8 months of age. By 9months of age, high ocular pressure has been ongoing in the eyes of themajority of the D2 mice. The D2 mice subsequently have a progressivevision loss, optic nerve damage, and inner retina dysfunction. At 12months of age, when designed experiments typically end, ˜70% of the D2mice eyes have a severe disease based on histological examination of theretina and optic nerve. glaucoma. Topical administration of compounds(e.g., memantine, timolol, or Latanoprost) lowers IOP in D2 mice andreduces the risk of developing neurodegeneration as they do in humanglaucoma. Control D2-Gpnmb⁺ is an age- and strain-matched mice that donot develop glaucoma.

Ocular Pressure in Glaucoma

Intraocular pressure (IOP) can be determined using, for example,Goldmann applanation tonometry (Haag Streit, Bern, Switzerland). Inhumans, normal IOP is 12-21 mmHg. IOP that exceeds 21 mmHg is consideredhigh. Elevated IOP is a major risk factor in glaucoma. Of those withPOAG (primary open angle glaucoma, the most common glaucoma accountingfor >90% of cases), 25 mmHg is the median untreated baseline IOP.

The Baltimore eye study (www.ncbi.nlm.nih.gov/pub/12049574) reportedthat high risk=IOP>25.75 mmHg; moderate risk=IOP 23.75-25.75 mmHg; andlow risk=IOP <23.75 mmHg. Lowering IOP by 20% to a level below 24 mmHgdecreases risk of progression from 9.5% to 4.4% at 5 years. The chanceof blindness in 1 eye is 27% after 10 years post diagnosis, and 38.1%after 20 years. The chance of blindness in both eyes is 6% and 13.5%respectably.

In human glaucoma patients, such as primary open agent glaucoma (POAG)patients, glaucoma is asynchronous and age-related (most commonlyoccurring at >40 years old). Untreated glaucoma takes on, average, 14years to progress from early to late stage disease when IOP is 21-25mmHg. This rate of progression rapidly increases as IOP increases (˜3years to progress from early to late stage disease when IOP>30 mmHg).

In human patients, treatments that lower IOP (surgical orpharmacological) reduce the risk of developing neurodegeneration. Therates of disease progression and percentage chance of going blind arelikely misrepresented due to patients not being diagnosed. It isgenerally believed that high IOP does not always indicate glaucoma (˜30%blind after 10 years, ˜40% after 20). Lowering IOP does not cureglaucoma, but does reduce the risk factor by 58%. Despite theconventional IOP lowering preventions, there is still a risk of visionloss, and even blindness. There are limited available neuroprotectivestrategies in glaucoma. Vision loss in glaucoma is irreversible. Infact, glaucoma is the leading case of irreversible blindness in theworld.

Retinal Ganglion Cells (RGCs)

Retinal ganglion cells (RGCs) are the output neuron of the retina. Theyreceive visual information from the photoreceptors (i.e., rods andcones) via intermediate neurons (i.e., bipolar cells and amacrinecells). This visual information starts as the photons in light, andculminates in an electric potential at retinal ganglion cell synapses.RGCs have long axons that leave the cell body and traverse across theretina to the optic disc (i.e., blind spot) where they exit out of theeye (optic nerve head). Just beyond the optic nerve head (myelintransition zone), retinal ganglion cell axons become myelinated and formthe optic nerve (i.e. the optic nerve is a bundle of retinal ganglioncell axons, in the mouse this is ˜50,000 depending on the strain). Axonsin the optic nerve eventually reach terminal visual centers in the brainthat then relay these signals on or process this information themselves.Two important visual centers that retinal ganglion cell axons terminatein are the lateral geniculate nucleus (LGN) and the superior colliculus(sup. col./SC).

Retinal ganglion cells are specifically affected (i.e., cell loss) inglaucoma. Damage to the RGCs likely occurs at the axon at the site ofoptic nerve head. It is speculated that because of the stress induced onthe eye by abnormally high IOP, the optic nerve head is a “weak spot” inthe eye where mechanical insults to the retinal ganglion cell axon mayoccur. The axon is not the only point of insult in the retinal ganglioncell, as pressure throughout the eye also must affect the soma anddendrites as well. The exact underlying mechanism of how RGCs damage isnot totally clear.

In the DBA/2J (D2) mice, the present inventors showed that attime-points where IOP is high, there is no detectable axon loss in theoptic nerve no ocular neurodegeneration). Surprisingly, the, presentinventors discovered that there are early mitochondrial and molecularchanges, dendrite atrophy, and synaptic loss. This finding suggests thatthe effects of IOP manifest in other compartments of the retinalganglion cell, not just the axon in the optic nerve.

Neurodegeneration Treatment

In certain embodiments, the agent comprises a nicotinamide adeninedinucleotide (NAD⁺) precursor (e.g., nicotinic acid, nicotinamide (NAM),nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or acombination thereof), a Krebs Cycle intermediate or precursor thereof(e.g., pyruvate), or a combination thereof.

In certain embodiments, the agent comprises a nicotinamide adeninedinucleotide (NAD⁺) precursor, e.g., nicotinic acid, nicotinamide (NAM),nicotinamide mononucleotide, nicotinamide riboside, or a combinationthereof. In certain embodiments, the NAD⁺ precursor is nicotinamide ornicotinamide riboside.

Nicotinic acid (also known as niacin, nicotinate, vitamin B3, andvitamin PP) is a vitamin. Its corresponding amide is called nicotinamideor niacinamide. These vitamins are not directly interconvertable;however, both nicotinate and nicotinamide are precursors in thesynthesis of redox. pairs NAD⁺/NADH (nicotinamide adenine dinucleotide)and NAD⁺/NADH (nicotinamide adenine dinucleotide phosphate). Thenicotinate and nicotinamide metabolic pathway can be referred to hereinas the NAD⁺ synthesis pathway.

NAD⁺ is synthesized through two metabolic pathways: a salvage pathwayand a de novo pathway. In the salvage pathway, NAD⁺ can be synthesizedfrom external sources of precursor compounds (e.g., nicotinic acid,nicotinamide, nicotinamide riboside, etc.). In the de novo pathway, NAD⁺can be synthesized from quinolinate produced during the metabolism ofamino acids (e.g., tryptophan, aspartate, etc.).

NAD⁺ precursor of the invention includes nicotinic: acid, nicotinamide,nicotinamide riboside, etc., as well as salts thereof, and analogsthereof. In certain embodiments, administering the NAD⁺ precursor of theinvention leads to increased intracellular level of NADt.

In certain embodiments, the agent comprises a Krebs Cycle intermediateor precursor thereof, or a combination thereof. For example, the Krebscycle intermediate or precursor is Oxaloacetate, Acetyl CoA, Citrate,CoA-SH, cis-Aconitate, D-Isocitrate, NAD⁺, Oxalosuccinate, NADH,α-Ketoglutarate, Succinyl-CoA, GDP, ubiquinone, Succinate, Fumarate,L-Malate, pyruvate, a monosaccharide (such as glucose, galactose,fructose), a disaccharide (such as sucrose, maltose, lactose), or acombination thereof.

Thus the invention provides pharmaceutical compositions that includenicotinic acid and/or nicotinamide riboside and/or nicotinamide and/ornicotinic acid metabolites. The nicotinic acid and/or nicotinamideriboside and/or nicotinamide and/or nicotinic acid metabolites can beused in free form. The term “free,” as used herein in reference to acomponent, indicates that the component is not incorporated into alarger molecular complex. In some embodiments, the nicotinic acid can becomprised in niacin. The nicotinic acid and/or nicotinamide ribosideand/or nicotinamide and/or nicotinic acid metabolites can be in a saltfor.

In some embodiments, any of the compositions described herein caninclude salts, derivatives, metabolites, catabolites, anabolites,precursors, and analogs thereof. For example, the metabolites caninclude nicotinyl CoA, nicotinuric acid, nicotinate mononucleotide,nicotinate adenine dinucleotide, or nicotinamide adenine dinucleotide.In some embodiments, the compositions comprise nicotinamide. In someembodiments, the compositions can be substantially free of nicotinicacid metabolites.

In certain embodiments, a salt is selected from the group consisting offluoride, chloride, bromide, iodide, formate, acetate, ascorbate,benzoate, carbonate, citrate, carbamate, formate, gluconate, lactate,methyl bromide, methyl sulfate, nitrate, phosphate, diphosphate,succinate, sulfate, and trifluoroacetate salt.

NAM/nicotinic acid analogs may also be used in the subject invention.Suitable analogues of nicotinic acid include, for example,isonicotinamide and N-methyl-nicotinamide.

In certain embodiments, keto-, ethyl-, benzyl-, or other non-saltembodiments of NAM can also be used in the instant invention.

The toxicity of NAM/nicotinamide/NAD/NR/pyruvate is low, and relativelylarge amounts may be administered without toxic effect.

In mice, the present compounds (e.g., NAM/nicotinamide/NAD/NR/pyruvate)can be administered with a daily dosage of about 200-1,000 mg/kg/day toprovide a neuroprotective effect. Preferably, the daily dosage is about40-600 mg/kg/day. More preferably, the daily dosage is about 550mg/kg/day. At these dosages, the present compounds afford aneuroprotective effect.

In mice, the present compounds (e.g., NAM/nicotinamide/NAD/NR/pyruvate)can be administered at a dose of about 1,000-5,000 mg/kg/day to providean IOP lowering effect. Preferably, the daily dosage is about1,500-3,000 mg/kg/day. More preferably, the daily dosage is about 2,000mg/kg/day. At these dosages, the present compounds afford an IOPlowering effect.

With regard to PQQ, it is tolerated at least at 2,000 mg/kg/day in mice.The neuroprotective effect provided by PQQ is about 20-2,000 mg/kg/day,about 100-1,000 mg/kg/day, or about 500 mg/kg/day in mice.

To convert doses of pharmaceutical composition expressed in terms ofmg/kg from one species (e.g., mouse) to an equivalent surface area doseexpressed as mg/kg in another species (e.g., human), the follow tableprovides the approximate factors for conversion, based on theassumptions and constants in Freireich et al., Quantitative comparisonof toxicity of anticancer agents in mouse, rat, dog, monkey and man,Cancer Chemother Rep. 50(4):219-244, 1966 (incorporated herein byreference).

Equivalent Surface Area Dosage Conversion Factors To Mouse Rat MonkeyDog Human (20 gram) (150 gram) (3 kg) (8 kg) (60 kg) From Mouse 1 ½ ¼ ⅙1/12 Rat 2 ½ ¼ 1/7 Monkey 4 2 1 ⅗ ⅓ Dog 6 4 1⅔ 1 ½ Human 12 7 3 2 1

Thus, a dose of 550 mg/kg in mouse is equivalent (assuming equivalencyon the basis of mg/m²) to 550 mg/kg×¼=137.5 mg/kg in monkey, and 550mg/kg× 1/12=45.8 mg/kg in a 60 kg human (or 2.85 g).

The somewhat simplified conversion factors in the table above are basedon the body weight to surface area ratio [km] for the respectivespecies. If more precise conversions are desired, the dosage conversioncan also be based on the km factors of the respective species listedbelow.

Representative Surface Area to Weight Ratios [km] for Various SpeciesSpecies Body Weight (kg) Surface Area (m²) km factor Mouse 0.02 0.00663.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 Dog* 8.0 0.40 20 Human (Child)20 0.8 25 Human (Adult) 60 1.6 37 *depending on specific dog breeds, thekm factor may be different. For example, in some terriers and otherbreeds prone to glaucoma, the km factor of about 10 may be used.

Thus, a dose of 550 mg/kg in mouse is equivalent to 550mg/kg×3.0/37=44.6 mg/kg in a 60 kg adult human (or 2.68 g), and 550mg/kg×3.0/25=66 mg/kg in a 20 kg human child (or 1.32 g).

The above table can also be used to express a mg/kg dose in any givenspecies as the equivalent mg/sq.m. dose, by multiplying the dose by theappropriate km factor. For example, in adult humans, 100 mg/kg isequivalent to 100 mg/kg×37 kg/sq.m.=3700 mg/sq.m.

In humans (18 60 kg individuals), the present compounds (e.g.,NAM/nicotinamide/NAD/NR/pyruvate) can be administered at a daily dosageof 0.5-10 grams to provide a neuroprotective effect. Preferably, thedaily dosage is about 1-5 grams/day. Preferably, the daily dosage isabout 2-4 grams/day. More preferably, the daily dosage is about 2.5grams/day. At these dosages, the present compounds afford aneuroprotective effect.

In humans (˜60 kg individuals), the present compounds (e.g.,NAM/nicotinamide/NAD/NR/pyruvate) can be administered at a daily dosageof about 5-25 grams/day to provide an IOP lowering effect. Preferably,the daily dosage is about 10-20 grams/day. Preferably, the daily dosageis about 8-15 grams/day. More preferably, the daily dosage is about 10grams/day. At these dosages, the present compounds afford an IOPlowering effect.

In humans (˜60 kg individuals), PQQ can be administered at a dailydosage of about 2-160 mg/kg/day, or about 10-100 mg/kg/day, or about 50mg/kg/day to afford a neuroprotective effect. In certain embodiments,PQQ can be administered at a daily dosage of ˜10 mg -10 g a day, about50 mg -1 g a day, or about 500 mg a day.

Ideally, typical dosing may be once, twice or three times a day. Totaldaily dose may be administered once, or administered as two or threeseparate doses (e.g., with each dose being ½or ⅓of the daily total). Formultiple dosing, each dose can be the same amount or different amounts.The pharmaceutical composition may be administered in the morning orevening. The pharmaceutical composition may be taken with or withoutmeals.

Therapeutic Agent to Treat Neurodegeneration Disorder

In one aspect, the present invention provides a method of treating orpreventing a neurodegenerative disorder in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of an agent that increases intracellular level of NADt,or that increases intracellular level of NAD⁺, NADH, GSH, GSSG,pyruvate, or PQQ.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of NAM,nicotinamide mononucleotide (NMN or β-NMN), NAD, pyruvate, PQQ orglutathione.

The agent used in the pharmaceutical composition is a neuroprotectiveagent to treat or prevent such neurodegenerative disorder, such asglaucoma.

Many different types of insults or neurodegenerative diseases orconditions can lead to neuronal damage or death, for example: metabolicstress caused by hypoxia, hypoglycemia, diabetes, loss of ionichomeostasis, physical injury of neurons, exposure to toxic agents andnumerous diseases affecting the nervous system including inherited ornon-inherited neurodegenerative disorders. In certain embodiments, theneurodegenerative disorder involves axon degeneration. In certainembodiments, the neurodegenerative disorder involves Walleriandegeneration or Wallerian-like degeneration. For example, the Walleriandegeneration may result from neuronal injury, such as injuries resultingfrom disease, trauma, or a chemotherapeutic agent.

It will be appreciated that this is only an illustrative list. Thepresence of an agent that is neuroprotective will enable a neuron toremain viable upon exposure to insults or disease conditions which maycause a loss of functional integrity in an unprotected neuron. Suchagents may also prevent the neuron from undergoing damage in response toinsults by making the neuron more resilient.

Pharmaceutical Composition

A pharmaceutical formulation comprises a pharmacologically activeingredient in a form not harmful to the subject it is being administeredto and additional constituents designed to stabilize the activeingredient and affect its absorption into the circulation or targettissue.

The pharmaceutical compositions according to the invention may beformulated with pharmaceutically acceptable carriers or diluents as wellas any other known adjuvants and excipients in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995.

Suitable pharmaceutical acceptable carriers include inert solid diluentsor fillers, sterile aqueous solutions and various organic solvents.Examples of solid carriers are lactose, terra alba, sucrose,cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate,stearic acid and lower alkyl ethers of cellulose. Examples of liquidcarriers are syrup, peanut oil, olive oil, phospholipids, fatty acids,fatty acid amines, polyoxyethylene and water.

Administration of the pharmaceutical composition according to theinvention may be through various routes, for example oral, parenteral(including subcutaneous, intramuscular, intradermal), intravitreal,intraocular (e.g., in the form of eye drops) and the like. In onepreferred embodiment, the pharmaceutical composition is for oraladministration (including administration of the pharmaceuticalcomposition as part of a drink). In one preferred embodiment, thepharmaceutical composition is for intraocular administration.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can he performed by means of an infusion pump. As a stillfurther option, the formulation of the invention can also be adapted totransdermal administration, e.g., by needle-free injection or from apatch, optionally an iontophoretic patch, or transmucosal, e.g., buccal,administration.

The pharmaceutical composition of the invention may be administered insuitable dosage forms, for example, as solutions, suspensions,emulsions, tablets, coated tablets, capsules, hard gelatine capsules andsoft gelatine capsules, drops, eye drops, ophthalmic ointments,ophthalmic rinses, injection solution, and the like.

The pharmaceutical composition of the invention may further becompounded in, or attached to, for example through covalent, hydrophobicand electrostatic interactions, a drug carrier, drug delivery system andadvanced drug delivery system in order to further enhance stability ofthe composition, increase bioavailability, increase solubility, decreaseadverse effects, achieve chronotherapy well known to those skilled inthe art, and increase patient compliance or any combination thereof.

Examples of carriers, drug delivery systems and advanced drug deliverysystems include, but are not limited to, polymers, for example celluloseand derivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylate,polymers, polylactic and poly glycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,1.2 phase and dispersions thereof, well known to those skilled in theart of phase behavior in lipid-water systems, polymeric micelles,multiple emulsions, self emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

The pharmaceutical composition of the current invention may be useful inthe composition of controlled, sustained, protracting, retarded, andslow release drug delivery systems. More specifically, but not limitedto, the pharmaceutical composition may be useful in the composition ofparenteral controlled release and sustained release systems (bothsystems leading to a many-fold reduction in number of administrations),well known to those skilled in the art. Even more preferably, arecontrolled release and sustained release systems administeredsubcutaneously. Without limiting the scope of the invention, examples ofuseful controlled release system and compositions are hydrogels,oleaginous gels, liquid crystals, polymeric micelles, microspheres andnanoparticles.

Neurodegeneration Diseases

The pharmaceutical compositions of the invention are predicted to be ofutility in the treatment of neurodegenerative disorders involving axondegeneration, such as Wallerian degeneration. Examples of disorderswhere such degeneration may be of importance include diabeticneuropathy, motor neuron disease, multiple sclerosis, peripheralneuropathy, stroke and other ischaemic disorders, traumatic brain injuryand the like. This list is for illustrative purposes only and is notlimiting or exhaustive.

In certain embodiments, the neurodegenerative disorder is one or more ofdiabetic neuropathy, traumatic brain injury, ischemia, peripheralneuropathy, or an ophthalmic disorder such as glaucoma. In certainembodiments, the neurodegenerative disorder is glaucoma.

In one embodiment the pharmaceutical composition is intended for use asa neuroprotective medicament in the treatment or prevention of aneurodegenerative disorder resulting from neuronal injury. In a furtherembodiment the modulator is intended for use as a neuroprotectivemedicament in the treatment of a neurodegenerative disorder involvingaxon degeneration (e.g., Wallerian degeneration) resulting from neuronalinjury. The term “injury” as used herein refers to damage inflicted onthe neuron, whether in the cell body or in axonal or dendriticprocesses. This can be a physical injury in the conventional sense,i.e., traumatic injury to the brain, spinal cord or peripheral nervescaused by an external force applied to a subject. Other damagingexternal factors are for example environmental toxins such as mercuryand other heavy metals, arsenic, pesticides and solvents. Alternatively,injury can result from an insult to the neuron originating from withinthe subject, for example: reduced oxygen and energy supply as inischemic stroke and diabetic neuropathy, autoimmune attack as inmultiple sclerosis or oxidative stress and free-radical generation as isbelieved to be important in amyotrophic lateral sclerosis, injury isalso used here to refer to any defect in the mechanism of axonaltransport.

In another embodiment, the subject pharmaceutical composition isintended for use as a neuroprotective medicament wherein theneurodegenerative disorder is caused by a neuronal injury resulting froma disease.

In one embodiment, the neuronal injury results from aging.

In one embodiment, the neuronal injury results from trauma. In oneembodiment, the disorder is a neuronal injury induced by achemotherapeutic agent. Certain drugs used in cancer chemotherapy suchas Taxol, Velcade and vincristine, cause peripheral neuropathy whichlimits the maximum doses at which they can be used. Recent studiessuggest that neurons suffering from Taxol or vincristine toxicityundergo Wallerian-like changes in their morphology and in the underlyingmolecular events. Inhibiting Wallerian degeneration could beparticularly effective in this condition as neurons are only temporarilyexposed to the neurotoxic agent. Simultaneous administration of Taxol orvincristine with an agent inhibiting Wallerian degeneration couldtherefore allow the drug to be used at substantially higher doses thanis currently possible, thus further combating the cancer.

Age is a common risk factor for most glaucoma, the present therapeuticmethods offers protection against age-related declines in NAD andprotect patients from developing glaucoma (i.e., preventingneurodegeneration). The present provides a combined therapy ofadministering NAM and/or pyruvate (to replenish NAD levels) as well asgene delivery of NMNAT1 gene. Given the surprising potency of NAM and/orpyruvate and gene therapy, the combined therapy is an attractiveapproach for treating and preventing glaucoma.

Gene Therapy to Treat Neurodegeneration

Gene therapy is an attractive method for overcoming compliance issuesand improving efficacy. The present invention provides a method of genetherapy to treat neurodegeneration. Axon degeneration is an area ofunmet therapeutic need. Neurodegeneration causes symptoms in motorneuron disease, glaucoma, Alzheimer's disease, and multiple sclerosis.In diabetes, it causes neuropathic pain and distal sensory loss, whichis a leading cause of limb amputation. It is also a dose-limiting sideeffect in cancer chemotherapy. Progressive axon degeneration due tostretch injury is the major pathology in traumatic brain injury, andfailure to protect white matter limits the treatment for stroke. Aroundhalf of the human population will eventually suffer one or more of thesedisorders, which significantly reduces quality of life.

In certain embodiments, the present invention provides a method oftreating neurodegeneration in the eye. The present invention provides amethod of replacing copies of dysfunctional genes to the eye. Genes areintroduced using viral gene delivery. In certain embodiments, thevectors include adenovirus, adeno-associated virus (AAV). In a preferredembodiment, the AAV is AAV2.2. For purposes of this application, it isintended to encompass other AAV serotypes including AAV1, AAV2, AAV4,AAV5, AAV8, AAV9, and the like. In other embodiments, the vector is aLentivirus, typically a pseudotype HIV-based vector.

To gene deliver targeting RGCs, viral vectors are introduced to thevitreal chamber. In a preferred embodiment, gene delivery is targeteddirectly proximal to the inner retina. Intravitreal injections areroutinely practiced in ophthalmic surgery and can be performed safely inan office/clinic location.

The present invention provides viral gene delivery to the eye as anattractive prospect as it allows a large number of cells to betransfected life-long, delivering a targeted gene product. To the bestof the inventors' knowledge, gene therapy in the eye represents a novelapproach of glaucoma treatment. Such gene therapy to increase NAD⁺applied to human complex diseases such as glaucoma represents a novelapproach of treatment.

In one aspect, the present invention provides a method of delivering agene to an afflicted eye whereby to enhance expression of NAD. Incertain embodiments, the gene therapy composition according to theinvention (e.g., a polynucleotide on a viral vector) is administeredintravitreally or intraocularly. In one preferred embodiment, the genetherapy is for intravitreal administration. It will be appreciated thatthe preferred route will depend on the general condition and age of thesubject to be treated, the nature of the condition to be treated and theactive ingredient chosen.

In one aspect, the present invention provides a gene therapy to delivera gene to an eye to increase the expression of Nmnat.

In certain embodiments, the gene that increases protein expression ofNmnat includes Nmnat-1, Nmnat-2, or Nmnat-3. In certain embodiments, thegene is Nmnat-1 (e.g., human NMNAT1).

NMNAT

Nicotinamide nucleotide adenylyltransferase 1 (Nmnat1 [mouse], NMNAT1[human]) encodes an enzyme which catalyzes a key step in thebiosynthesis of nicotinamide adenine dinucleotide (NAD). The encodedenzyme is one of several nicotinamide nucleotide adenylyltransferases,and is specifically localized to the cell nucleus. Alternative splicingof this gene results in multiple (at least three) transcript variants.

The NCBI reference sequences (RefSeq) for human NMNAT1 isoform (1)includes: NM_022787.3 (nucleotide) and NP_073624.2 (protein), thenucleotide sequence of which is known as transcription variant (1) thatencodes the longer NMNAT1 isoform (I); and NM_001297778.1 (nucleotide)and NP_001284707.1 (protein), the nucleotide sequence of which is knownas transcript variant (2) that differs in the 5′ UTR region whencompared to variant 1. Variants 1 and 2 encode the same isoform (1), andcan all be used in the methods of the invention. All sequences areincorporated by reference.

The related nicotinamide nucleotide adenylyltransferase 2 (nmnat2),unlike the other human family members localized to the nucleus andubiquitously expressed, this enzyme is cytoplasmic, and is predominantlyexpressed in the brain. Alternative splicing of this gene results in twotranscript variants.

The NCBI reference sequences (RefSeq) for human NMNAT2 isoform (1)includes: NM_015039.3 (nucleotide) and NP_055854.1 (protein), thenucleotide sequence of which may be used in the method of the invention.All sequences are incorporated by reference.

The related nicotinamide nucleotide adenylyltransferase 3 (nmnat3)encodes a protein localized to mitochondria, and may also play aneuroprotective role as a molecular chaperone. Alternatively splicedtranscript variants (at least 6) encoding multiple isoforms (at least 5)have been observed for this gene.

The NCBI reference sequences (RefSeq) for human NMNAT3 isoform (3)includes: NM_001320510.1 (nucleotide) and NP_001307439.1 (protein), thenucleotide sequence of which is transcription variant 3 that encodes thelongest isoform (3), and may be used in the method of the invention. Allsequences are incorporated by reference.

NMNAT2 is emerging as an important NAD producing enzyme in axons andprotects from axon degeneration. Ongoing stress negatively impactsNmnat2 expression in RGCs (q<0.05 in D2 glaucoma Group 4, the finalstage detected before glaucomatous degeneration). This decline may beimportant in the transition to axon degeneration in glaucoma. NMNAT2expression is decreased in brains with Alzheimer's disease and has ahighly variable depression in aged postmortem human brains, which maycontribute to the variable vulnerability to these conditions.

Numerous related nmnat sequences in different species of animals (suchas house mouse Mus musculus) are readily available from publicdatabases, such as NCBI RefSeq, GenBank, etc. All sequences areincorporated herein by reference.

It is believed that gene therapy employing NAMPT will lead to axon celltoxicity, albeit elevating the NAD⁺ levels in the cells. NAMPT functionsto convert NAM to NMN, which is then converted to NAD⁺. IntracellularNAD⁺ is a key molecule associated with axon degeneration. When NADlevels are low, axons rapidly degenerate. It is also established thatfollowing axonal injury in sciatic nerve axons, NMN accumulates rapidly.In this model axon degeneration, NMN levels rise within 12 hrs, followedby neural injury occurs at 36-hr following injury. Blocking the NMNproducing enzyme NAMPT using FK866 potentially inhibits this axondegeneration, hinting that NMN is toxic to neurons. Axon degenerationappears to be NMN-dependent, and inhibiting the increase of NMN usingFK866 protects against axon degeneration. In a zebrafish model of axondegeneration (two-photon-laser axotomy), FK866 potently delayed axondegeneration. In a cell culture model of neurite degeneration (superiacervical ganglion; SCG), rapidly clearing NMN (by overexpression of thebacterial enzyme NMN deamidase which converts NMN to NAMN) robustlyprotects from axon degeneration.

Accordingly, the present invention represents an unexpected finding thatgene therapy employing NMNAT, unlike that of NAMPT, is effective inprotecting ocular neurodegeneration.

Thus we chose Nmnat1 over Nampt, as Nampt overexpression would drive theproduction of NMN, which, without proper clearance, is toxic to neurons.

Wld^(S)

Axonal degeneration is a common component of neurodegenerative disease.There are two models that attempt to explain this greater degree ofdistal axonal degeneration. The first is “dying back” in whichdegeneration spreads retrogradely from the nerve terminals. The secondis Wallerian degeneration, where degeneration spreads from the site of alesion in either direction according to the lesion type, ultimatelyresulting in loss of the axon distal to the lesion site, and leaving theproximal portion intact. Although strictly speaking, Walleriandegeneration only occurs in response to physical injury of the axon,similar mechanisms operate in diseases where no such injury hasoccurred. The latter is referred to as “Wallerian-like” degeneration.Both types of degeneration will hereinafter be jointly referred to as“Wallerian degeneration.”

The recently discovered Wld^(S) mouse has led to progress in theunderstanding of these two processes. In these animals, Walleriandegeneration occurs at a rate roughly ten times slower than in wild-typeanimals. Studies have shown that this mutation also delays pathologiesbelieved to involve “dying back” of axonal terminals. The Wld^(S) genetherefore provides a mechanistic link between the two models of axonaldegeneration.

Despite the identification and characterization of the Wld^(S) gene,progress towards understanding of the molecular trigger for Walleriandegeneration has been limited. Knowledge of this trigger could have aprofound impact on the understanding of the early stages of “dying-back”neurodegenerative diseases.

Mice with the Wld^(S) gene have delayed Wallerian degeneration. TheWld^(S) mutation is an autosomal-dominant mutation occurring in themouse chromosome 4. The gene mutation is a naturally occurring 85-kbtandem triplication, resulting in a mutated region containing twoassociated genes: nicotinamide mononucleotide adenylyl transferase 1(Nmnat-1) and ubiquitination factor e4b (Ube4b), and a linker regionencoding 18 amino acids. The protein created localizes within thenucleus and is undetectable in axons.

The mutation appears to cause no harm to the mouse. The only knowneffect is that the Wallerian degeneration is delayed by up to threeweeks on average after injury of a nerve. Recent studies suggest thatthe mutation protects axons by a poorly understood mechanism. While notwishing to be bound by any particular theory, it is likely that theWld^(S) mutation leads to overexpression and/or improved localization ofthe Nmnat (e.g., Nmnat-1) protein and increased NAD synthesis.

In certain embodiments, the method comprises administering apolynucleotide encoding Nmnat (e.g.,Nmnat-1, -2, or -3) or Wld^(S) tothe subject.

In certain embodiments, the polynucleotide encodes Wld^(s)or a humansequence equivalent thereof.

In certain embodiments, the method comprises administering NAM or aprecursor, and a polynucleotide encoding Wld^(S) to the subject.

In certain embodiments, the polynucleotide is administered to thesubject locally. For example, to treat glaucoma, the agent may belocally delivered to the affected eye(s),

Vectors

In certain embodiments, the polynucleotide is administered to thesubject on a viral vector. Exemplary suitable viral vector includes, butnot limited to, an AAV vector, an adenoviral vector, a Lentiviralvector, a retroviral vector, and the like. Preferably, the viral vectoris a AAV vector or a Lentiviral vector.

For instance, the polynucleotide may be introduced into the subject asexogenous genetic materials that may modulate the expression of one ormore target genes of interest. Such kind of gene therapy can be used,for example, in a method directed at repairing damaged or diseasedtissue, such as neuronal tissue. In brief, any suitable vectors,including an adenoviral, a lentiviral, or retroviral gene deliveryvehicle (see below), may be used to deliver genetic information, likeDNA and/or RNA to the subject. A skilled person can replace or repairparticular genes targeted in gene therapy. For example, a normal genemay be inserted into a nonspecific location within the genome of adiseased cell to replace a nonfunctional gene. In another example, anabnormal gene sequence can be replaced for a normal gene sequencethrough homologous recombination. Alternatively, selective reversemutation can return a gene to its normal function. A further example isaltering the regulation (the degree to which a gene is turned on or offof a particular gene. In certain embodiments, the target cells (such asneuronal stem cells) are ex vivo treated by a gene therapy approach andare subsequently transferred to the mammal, preferably a human being inneed of treatment.

Any art recognized methods for genetic manipulation may be used,including transfection and infection (e.g., by a viral vector) byvarious types of nucleic acid constructs.

For example, heterologous nucleic acids (e.g., DNA) can be introducedinto the subject by way of physical treatment (e.g., electroporation,sonoporation, optical transfection, protoplast fusion, impalefection,hydrodynamic delivery, nanoparticles, magnetofection), using chemicalmaterials or biological vectors (viruses). Chemical-based transfectioncan be based on calcium phosphate, cyclodextrin, polymers (e.g.,cationic polymers such as DEAE-dextran or polyethylenimine), highlybranched organic compounds such as dendrimers, liposomes (such ascationic liposomes, lipofection such as lipofection using Lipofectamine,etc.), or nanoparticles (with or without chemical or viralfunctionalization).

A nucleic acid construct comprises a nucleic acid molecule of interest,and is generally capable of directing the expression of the nucleic acidmolecule of interest in the cells into which it has been introduced.

In certain embodiments, the nucleic acid construct is an expressionvector wherein a nucleic acid molecule encoding a gene product, such asa polypeptide or a nucleic acid that antagonizes the expression of apolypeptide (e.g., an siRNA, miRNA, shRNA, antisense sequence, aptamer,ribozyme, antagomir, RNA sponge, etc.) is operably linked to a promotercapable of directing expression of the nucleic acid molecule in thetarget cells.

A DNA construct prepared for introduction into a particular celltypically includes a replication system recognized by the cell, anintended DNA segment encoding a desired polypeptide, and transcriptionaland translational initiation and termination regulatory sequencesoperably linked to the polypeptide-encoding segment. A DNA segment is“operably linked” when it is placed into a functional relationship withanother DNA segment. For example, a promoter or enhancer is operablylinked to a coding sequence if it stimulates the transcription of thesequence. DNA for a signal sequence is operably linked to DNA encoding apolypeptide if it is expressed as a pre-protein that participates in thesecretion of a polypeptide. Generally, a DNA sequence that is operablylinked are contiguous, and, in the case of a signal sequence, bothcontiguous and in reading phase. However, enhancers need not becontiguous with a coding sequence whose transcription they control.Linking is accomplished by ligation at convenient restriction sites orat adapters or linkers inserted in lieu thereof.

The selection of an appropriate promoter sequence generally depends uponthe host cell selected for the expression of a DNA segment. Examples ofsuitable promoter sequences include eukaryotic promoters well known inthe art (see, e.g., Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Third Edition, 2001). A transcriptional regulatorysequence typically includes a heterologous enhancer or promoter that isrecognized by the cell. Suitable promoters include the CMV promoter. Anexpression vector includes the replication system and transcriptionaland translational regulatory sequences together with the insertion sitefor the polypeptide encoding segment can be employed. Examples ofworkable combinations of cell lines and expression vectors are describedin Sambrook and Russell (2001, supra) and in Metzger et al. (1988)Nature, 334: 31-36.

Some aspects of the invention concern the use of a nucleic acidconstruct or expression vector comprising a nucleotide sequence asdefined above, wherein the vector is a vector that is suitable for genetherapy. Vectors that are suitable for gene therapy are known in theart, such as those described in Anderson (Nature, 392: 25-30, 1998);Walther and Stein (Drugs, 60: 249-71, 2000); Kay et al. (Nat. Med., 7:33-40, 2001); Russell (J. Gen. Virol., 81:2573-604, 2000); Amado andChen (Science 285:674-6, 1999); Federico (Curr. Opin. Biotechnol.,10:448-53, 1999); Vigna and Naldini (J. Gene Med., 2:308-16, 2000);Marin et al. (Mol. Med Today, 3:396-403, 1997); Peng and Russell (Curr.Opin. Biotechnol., 10:454-7, 1999); Sommerfelt (J. Gen. Virol.,80:3049-64, 1999); Reiser (Gene Ther., 7: 910-3, 2000); and referencescited therein (all incorporated by reference). Examples includeintegrative and non-integrative vectors such as those based onretroviruses, adenoviruses (AdV), adeno-associated viruses (AAV),lentiviruses, pox viruses, alphaviruses, and herpes viruses.

A particularly suitable gene therapy vector includes an Adenoviral (Ad)and Adeno-associated virus (AAV) vector, These vectors infect a widenumber of dividing and non-dividing cell types. In addition, adenoviralvectors are capable of high levels of transgene expression. However,because of the episomal nature of the adenoviral and AAV vectors aftercell entry, these viral vectors are most suited for therapeuticapplications requiring only transient expression of the transgene(Russell, J. Gen. Virol., 81:2573-2604, 2000; Goncalves, Virol 2(1):43,2005) as indicated above. Preferred adenoviral vectors are modified toreduce the host response as reviewed by Russell (2000, supra). Safetyand efficacy of AAV gene transfer has been extensively studied in humanswith encouraging results in the liver, muscle, CNS, and retina (Manno etal., Nat. Medicine, 2006; Stroes et al., ATVB, 2008; Kaplitt, Feigin,Lancet, 2009; Maguire, Simonelli et al. NEJM, 2008; Bainbridge et al.,NEJM, 2008).

AAV2 is the best characterized serotype for gene transfer studies bothin humans and experimental models. AAV2 presents natural tropism towardsskeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.Other examples of adeno-associated virus-based non-integrative vectorsinclude AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 andpseudotyped AAV. The use of non-human serotypes, like AAV8 and AAV9,might be useful to overcome these immunological responses in subjects,and clinical trials have just commenced (ClinicalTrials dot govIdentifier: NCT00979238). For gene transfer into a liver cell, anadenovirus serotype 5 or an AAV serotype 2, 7 or 8 have been shown to beeffective vectors and therefore a preferred Ad or AAV serotype (Gao,Molecular Therapy, 13:77-87, 2006).

An exemplary retroviral vector for application in the present inventionis a lentiviral based expression construct. Lentiviral vectors have theunique ability to infect non-dividing cells (Amado and Chen, Science285:674-676, 1999). Methods for the construction and use of lentiviralbased expression constructs are described in U.S. Pat. Nos, 6,165,782,6,207,455, 6,218,181, 6,277,633, and 6,323,031, and in Federico (Curr.Opin. Biotechnol. 10:448-53, 1999) and Vigna et at (J. Gene Med.2:308-16, 2000).

Generally, gene therapy vectors will be as the expression vectorsdescribed above in the sense that they comprise a nucleotide sequenceencoding a gene product (e.g., a polypeptide) of the invention to beexpressed, whereby a nucleotide sequence is operably linked to theappropriate regulatory sequences as indicated above. Such regulatorysequence will at least comprise a promoter sequence. Suitable promotersfor expression of a nucleotide sequence encoding a polypeptide from genetherapy vectors include, e.g., cytomegalovirus (CMV) intermediate earlypromoter, viral long terminal repeat promoters (LTRs), such as thosefrom murine Moloney leukaemia virus (MMLV) roes sarcoma virus, or HTLV-1, the simian virus 40 (SV 40) early promoter and the herpes simplexvirus thymidine kinase promoter. Additional suitable promoters aredescribed below.

Several inducible promoter systems have been described that may beinduced by the administration of small organic or inorganic compounds.Such inducible promoters include those controlled by heavy metals, suchas the metallothionine promoter (Brinster et al., Nature, 296:39-42,1982; Mayo et al., Cell, 29:99-108, 1982), RU-486 (a progesteroneantagonist) (Wang et al., Proc. Natl. Acad Sci. USA, 91:8180-8184,1994), steroids (Mader and White, Proc. Natl. Acad. Sci. USA,90:5603-5607, 1993), tetracycline (Gossen and Bujard, Proc. Natl. Acad.Sci. USA, 89:5547-5551, 1992; U.S. Pat. No. 5,464,758; Furth et al.,Proc. Natl. Acad. Sci. USA, 91:9302-9306,1994; Howe et al., J. Biol.Chem., 270:14168-14174, 1995; Resnitzky et al., Mol. Cell. Biol.,14:1669-1679, 1994; Shocked et al., Proc. Natl. Acad. Sci. USA,92:6522-6526, 1995) and the tTAER system that is based on themulti-chimeric transactivator composed of a tetR polypeptide, asactivation domain of VP 16, and a ligand binding domain of an estrogenreceptor (Yee et al., 2002, U.S. Pat. No. 6,432,705).

Suitable promoters for nucleotide sequences encoding small RNAs forknock down of specific genes by RNA interference (see below) include, inaddition to the above mentioned polymerase II promoters, polymerase IIIpromoters. The RNA polymerase III (pol III) is responsible for thesynthesis of a large variety of small nuclear and cytoplasmic non-codingRNAs including 5S, U6, adenovirus VA1, Vault, telomerase RNA, and tRNAs.The promoter structures of a large number of genes encoding these RNAshave been determined and it has been found that RNA pol III promotersfall into three types of structures (for a review see Geiduschek andTocchini-Valentini, Annu. Rev. Biochem., 57: 873-914, 1988; Willis, Eur.J. Biochem., 212:1-11, 1993; Hernandez, J. Biol. Chem., 276:26733-36,2001). Particularly suitable for expression of siRNAs are the type 3 ofthe RNA pol III promoters, whereby transcription is driven by cis-actingelements found only in the 5′-flanking region, i.e., upstream of thetranscription start site. Upstream sequence elements include atraditional TATA box (Mattaj el al., Cell, 55:435-442, 1988), proximalsequence element and a distal sequence element (DSE; Gupta and Reddy,Nucleic Acids Res., 19:2073-2075, 1991). Examples of genes under thecontrol of the type 3 pol III promoter are U6 small nuclear RNA (U6snRNA), 7SK, Y, MRP, HI and telomerase RNA genes (see, e.g., Myslinskiet al., Nucl. Acids Res., 21:2502-09, 2001).

A gene therapy vector may optionally comprise a second or one or morefurther nucleotide sequence coding for a second or further polypeptide.A second or further polypeptide may be a (selectable) marker polypeptidethat allows for the identification, selection and/or screening for cellscontaining the expression construct. Suitable marker proteins for thispurpose are, e.g., the fluorescent protein GFP, and the selectablemarker genes HSV thymidine kinase (for selection on HAT medium),bacterial hygromycin B phosphotransferase (for selection on hygromycinB), Tn5 aminoglycoside phosphotransferase (for selection on G418), anddihydrofolate reductase (DHFR) (for selection on methotrexate), CD20,the low affinity nerve growth factor gene. Sources for obtaining thesemarker genes and methods for their use are provided in Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York,2001.

Alternatively, a second or further nucleotide sequence may encode apolypeptide that provides for fail-safe mechanism that allows a subjectfrom the transgenic cells to be cured, if deemed necessary. Such anucleotide sequence, often referred to as a suicide gene, encodes apolypeptide that is capable of converting a prodrug into a toxicsubstance that is capable of killing the transgenic cells in which thepolypeptide is expressed. Suitable examples of such suicide genesinclude, e.g., the E. coil cytosine deaminase gene or one of thethymidine kinase genes from Herpes Simplex Virus, Cytomegalovirus andVaricella-Zoster virus, in which case ganciclovir may be used as prodrugto kill the IL-10 transgenic cells in the subject (see, e.g., Clair etal., Antimicrob. Agents Chemother., 31:844-849, 1987).

Administration Routes for Gene Therapy in the Eye

In one aspect, the present invention provides a method of gene therapyto treat ocular degeneration diseases. One skilled in the art wouldrecognize the routes for gene therapy in the eye that involvesintravitreal administration or intraocular administration.

Intraocular administration is delivering a gene composition anywhereinto eyeball.

In a preferred embodiment, the gene composition is injectedintravitreally. Intravitreal administration is delivering a genecomposition directly into the vitreous compartment of the eye, i.e., thefluid that makes up the posterior part of the eye, directly adjacent tothe retina. In certain embodiments, vitreous is removed to make spacefor drug during injection.

Intravitreal injections typically take place in the doctor's office, butcan also take place in the Operation Rooms in a hospital. Patientsgenerally undergo topical anesthesia (usually eye-drops orsoaked-sponge/cotton swabs), with commonplace drugs such asproparacaine, lidocaine, and the like.

In certain embodiments, the gene composition is injected subretinally(i.e., under the retina).

For injection, needle size of 27-32 gauge may be used. In a preferredembodiment, the needle size is 30-gauge. Needle injection is becoming apopular technique to inject agents into the eye and does not pose anyrisks. Injection can be deep or superficial. Injections may he straight(i.e. at 90 degrees to the surface of the eye), oblique (45-60 degrees),or double-pane (in oblique, out straight).

Gene Therapy in the Eye

The present invention provides a gene therapy using a gene compositionto elevate NAD⁺ in the RGCs. The eye is an ideal organ to utilize genetherapy, because it is small in size (i.e. less cells to transfect),easily accessible, partially/mostly immune privileged (i.e. small/littlechance of eliciting an immune response), and the fellow eye serves as acontra-lateral control and a backup.

Several eye-based gene therapy clinical trials have been underway forblinding disorders, For example, gene therapy of RPE65 gene via AAV2vector to treat Leber's Congenital Amaurosis is in Phase III clinicaltrial (NCT00481546, NCT00516477, NCT00643747 and NCT00999609); genetherapy of MERTK gene using AAV2 vector to treat Retinitis Pigmentosa isin Phase I (NCT014822195); gene therapy of ABCA4 gene using EIAVlentivirus vector to treat Stargardt's is presently in Phase II(NCT01367444); gene therapy of gene REP-1 gene using AAV2 vector totreat Chorioderemia is presently in Phase II (NCT02553135). The clinicalstudies in several clinics demonstrate that gene delivery to eye appearsto be safely tolerated.

Combined Therapy-An Additional Therapeutic Agent

In certain embodiments, the present invention provides a method ofcombined therapy treatment of neurodegeneration by administering to ahuman in need thereof a compound that enhances the intracellular levelof NAD⁺ in a nerve cell (e.g., RGC) and an additional therapeutic agent.

In certain embodiments, the additional therapeutic agent of theinvention for treating glaucoma includes, but not limited to an agentthat lowers IOP. Exemplary additional therapeutic agent includes, butnot limited to a beta blocker (such as Timolol maleate, Timololhemihydrate, Levobunolol HCL, Metipranolol Carteolol, Betaxolol and thelike), a non-selective adrenergic agonist (such as Epinepherine,Dipivefrin HCL), a selective α-2 adrenergic agonists (such asApraclonidine HCL, Brimonidine tartrate, and Brimonidine tartrate inPurite), or a Carbonic Anhydrase Inhibitor (CAI, such as acetazolamide(oral), acetazolamide (parenteral), methazolamide (oral), dorzolamide(topical), and brinzolamide (topical)).

In certain embodiments, the additional therapeutic agent includes aprostaglandin analog (such as Latanoprost, Travaprost, and Bimataprost(prostamide)), parasympathomimetic agonist (including direct cholinergicagonist such as pilocarpine HCL; and indirect cholinergic agents such asechothiophate iodide, demercarium iodide, and physostigmineisofluorophate), and carbachol (a mixed direct agonist/acetylcholinereleasing agent).

In certain embodiment, the additional therapeutic agent comprises afixed-combination medication that offers the potential advantage ofincreased convenience, compliance, efficacy, and cost. Thefixed-combination may comprise a topical beta-blocker combined with aprostaglandin analogue, an alpha-adrenoceptor agonist, or a topicalcarbonic anhydrase inhibitor. Exemplary fixed combination include: (1)dorzolamide and timolol, such as Dorzolamide hydrochloride 2% andtimolol maleate ophthalmic solution 0.5% (e.g., Cosopt, now available asgeneric), (2) brimonidine with timolol or brinzolamide, such asbrimonidine tartrate 0.2%, timolol maleate ophthalmic solution 0.5%(e.g., Combigan) and brimonidine tartrat 0.2% and brinzolamide 1% (e.g.,Simbrinza), or (3) latanoprost and timolol.

In certain embodiment, the additional therapeutic agent comprises ahyperosmotic agent such as oral glycerine, oral isosorbide, andintravenous mannitol that can rapidly lower IOP by decreasing vitreousvolume. They do not cross the blood-ocular barrier and therefore exertoncotic pressure that dehydrates the vitreous. The hyperosmotic agent istypically used in acute situations to temporarily reduce high IOP untilmore definitive treatments can be rendered.

The invention is illustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of Glaucoma Susceptible Genes/Pathways

(A) Selection of Mouse Model Mimicking Neurodegeneration in Humans

We chose the DBA/2J D2 (D2) mouse model to examine the pathogenesis ofneurodegeneration in humans. The D2 mouse is a widely used model ofglaucoma, which recapitulates the hallmark features of human glaucoma.In D2 mice mutant alleles of two genes (Gpnmb^(R250X), Tyrp1^(b)) causea progressive iris disease. This iris disease has two main components:and iris stromal atrophy (highly resembles essential iris atrophy inhumans) and iris pigment dispersion phenotypes (highly resemble pigmentdispersion syndrome in humans). Both essential iris atrophy and pigmentdispersion syndrome induce IOP) elevation and glaucoma in humans. Irisdisease in D2 eyes results in age-related elevated intraocular pressure(IOP) in the majority of eyes by 8-9 months of age. Optic nervedegeneration is almost complete by 12 months of age (typically >70%nerves have severe damage).

In this study, the D2 mice were verified that they undergo ocularneurodegeneration following age-related IOP increases. Specifically, weused D2 mice at 3 ages; namely, 4-, 9-, and 12-months of age.

(i) 4-month old D2 mice were verified to exhibit normal intraocularpressure (IOP) (i.e., 10-13 mmHg) and no glaucoma (i.e., these mice wereat an age preceding the development of IOP and pre-glaucoma; nodetectable neurodegeneration). At this age, D2 mice areindistinguishable from control mice.

(ii) 9-month old D2 mice were verified to exhibit high IOP (i.e., >21mmHg) but no neurodegeneration. Although conventional glaucoma is absentat this developmental stage (i.e., pre-neurodegeneration), eyes in the9-month old D2 mice undergo molecular changes defined as having “earlyglaucoma” herein.

(iii) 12-month old D2 mice were verified to exhibit variable high IOP(i.e., 14 to >21 mmHg) and neurodegeneration becomes apparent (i.e.,glaucoma; >60% eyes have severe neurodegeneration).

(iv) Control 02-Gpnmb⁺ mice were used in this study, because these miceexhibit neither high IOP nor neurodegeneration with aging. These miceare identical to D2 mice except for a correction of the iris diseasecausing Gpnmb^(R250X) mutation.

(B) Isolation of Retinal Ganglion Cells (RGC) from Retinas of D2 Mice

We harvested retina from eyes obtained from (i) 4-month old DBA/2J D2mice. (ii) 9-month old DBA/2J D2 mice, and (iii) age-, sex-, andstrain-matched 02-Gpnmb⁺ wildtype controls.

Retinal samples were first stained with an antibody cocktail. Retinalganglion cells (RGCs) were identified as Thy1.2⁺ cells (and negative forCd11b⁻, Cd11c⁻, Cd31⁻, Cd34⁻, Cd45.2⁻, GFAP⁻, DAPI⁻). FACS positive RCGswere plated and stained with SNAP-25 and β-tubulin (specific markers ofRGCs) to confirm the RGC status.

Using fluorescence-activated cell sorting (FAC sorting), we thenisolated RGCs from the freshly harvested retinas in these three groupsof mice. See FIGS. 27A-27E.

(C) RNA Sequencing

To identify susceptible genes/pathways leading; to glaucoma, weperformed RNA-sequencing (RNA-se) from RNA of RGCs obtained above in (B)to elucidate very early molecular changes within the RGCs that precedeneurodegeneration. Amplified dscDNA libraries (double-stranded copy DNA)generated from RNA and read at a depth of 35 million reads per sample.Data analysis was performed at a false discovery rate (FDR, q) ofq<0.05. Samples from all groups (B) were successfully amplified andsequenced.

We performed additional metabolic profiling of neural retinas from 4-,9-, and 12-months old D2 and D2-Gpnmb⁺ eyes. Metabolic profiling wasperformed using targeted assays following the manufacturersrecommendations. The following metabolites were profiled: NAD+/NADH(i.e. total NAD, NAD(t)), GSH/GSSG (i.e. total glutathione,glutathione(t)), and pyruvate.

(D) Hierarchical Clustering (HC)

In this study, we sequenced the RNA from the isolated RGCs at a depth of35 million reads per sample. Unsupervised hierarchical clustering (MC)was used to define molecularly determined stages of glaucoma amongsamples that are at initial stages of disease and morphologicallyindistinguishable from age-matched D2-Gpnmb⁺ or young controls.

HC identified 4 distinct groups of 9-month old D2 samples (i.e., Group1, Group 2, Group 3 and Group 4). Group 1 clustered with all of thecontrol samples and represents D2 RGCs with no detectable glaucoma at amolecular level. All samples in Groups 2 to 4 were at early stages ofdisease, increasing group number were observed reflecting an increasingdistance from controls (greater disease progression at a transcriptomiclevel) (FIGS. 1, 2A, and 2B).

As disease progressed, there was an increase in transcript abundancethat was most pronounced for mitochondrial reads (FIG. 3A). Imbalancesin the relative proportions of mitochondrial molecules encoded bynuclear and mitochondrial genomes negatively impact mitochondrialfunction. In D2 Groups 2 to 4, differential expression of genes encodingmitochondrial proteins, and significant enrichment of differentiallyexpressed genes (compared to age- and sex-matched D2-Gpnmb⁺ controlRGCS) in the mitochondrial dysfunction and oxidative phosphorylationpathways further point to mitochondrial abnormalities within RGCs (FIGS.3A-3J, and 3K, and Tables 1-3 below).

Total read abundance across the transcriptome (i.e. all transcribedgenes) was split into those reads derived from the nuclear transcriptome(i.e. encoded in the nucleus) or the mitochondrial transcriptome (i.e.encoded at the mitochondrion). Read abundance increased in bothtranscriptomes in D2 RGCs but was most abundant in themitochondria-derived reads, indicating an imbalance that would favormitochondrial dysfunction (FIG. 3A).

Top enriched pathways (IPA) that appear in D2 Groups 2, 3, and 4. Thereare no enriched pathways in D2 Group 1 as there is only 1 differentiallyexpressed (DE) gene. This D2 Group 1 represents eyes that have notundergone glaucomatous insults (FIG. 3B).

We prepared a plot of all mitochondrial proteins encoded by nuclear (andnot mitochondrial) genes. DE genes are shown in red. Non-DE genes areshown in grey. The increasing abundance of nuclear derived transcriptsencoding mitochondrial proteins further indicates an imbalance inmitochondrial turnover or function (FIG. 3C).

An increase in expression of mitochondrial fission genes (Dnm1 and Fis1)indicates a pro-fission event in mitochondria early in glaucoma.Increase fission is associated with increased mitochondrial turnover anddisease. Mutations that affect mitochondrial fusion/fission dynamicstypically are lethal or cause neurological conditions (including, butnot limited to, dominant optic atrophy, Charcot-Marie-Tooth disease)(FIG. 3D).

There was an increased in expression of genes involved in themitochondrial unfolded protein response. This is a stress responsewithin cells that typically often precedes apoptosis (programmed celldeath) (FIG. 3E).

Individual plots of genes in mitochondria-relevant pathways wasprepared. DE genes are shown in red, non-DE genes are shown in grey.Note the upregulation of mitochondrial genes across the pathways,especially in oxidative phosphorylation and reactive oxygen speciesmetabolism, which points to an energy crisis within the cell (FIG, 3F).

The protein analysis (Western blot) of the mitochondrial pro-apoptoticmolecule cytochrome C from the retina shows that cytochrome C wasupregulated at both the gene and the protein levels during earlyglaucoma (9 month) and was significantly upregulated by 12 month (FIGS.3G and 3I).

Protein analysis also confirms that there was an eIF2 upregulationwithin the retina (FIGS. 3H and 3J).

TABLE 1 Mitochondrial complex genes list from FIGS. 1, 3A-3E, 3K 4A, and4B Complex Gene Complex Gene I ENSMUSG00000064341 I ENSMUSG00000064345 IENSMUSG00000064360 I ENSMUSG00000064363 I ENSMUSG00000064367 IENSMUSG00000064368 I ENSMUSG00000065947 I Ndufa1 I Ndufa10 I Ndufa11 INdufa12 I Ndufa13 I Ndufa2 I Ndufa3 I Ndufa4 I Ndufa412 I Ndufa5 INdufa6 I Ndufa7 I Ndufa8 I Ndufa9 I Ndufab1 I Ndufaf1 I Ndufaf2 INdufaf3 I Ndufaf4 I Ndufaf5 I Ndufaf6 I Ndufaf7 I Ndufb10 I Ndufb11 INdufb2 I Ndufb3 I Ndufb4 I Ndufb5 I Ndufb6 I Ndufb7 I Ndufb8 I Ndufb9 INdufc1 I Ndufc2 I Ndufs1 I Ndufs2 I Ndufs3 I Ndufs4 I Ndufs6 I Ndufs7 INdufs8 I Ndufv1 I Ndufv2 I Ndufv3 II Sdha II Sdhb II Sdhc II Sdhd IIICyc1 III ENSMUSG00000064370 III Uqcr10 III Uqcr11 III Uqerb III Uqcrc1III Uqcrc2 III Uqcrfs1 III Uqcrh III Uqcrq IV Cox4i1 IV Cox4i2 IV Cox5aIV Cox5b IV Cox6a1 IV Cox6b1 IV Cox6b2 IV Cox6c IV Cox7a2 IV Cox7a21 IVCox7b IV Cox7c IV Cox8a IV ENSMUSG00000064351 IV ENSMUSG00000064354 IVENSMUSG00000064358 V Atp5a1 V Atp5b V Atp5c1 V Atp5d V Atp5e V Atp5f1 VAtp5g1 V Atp5g2 V Atp5g3 V Atp5h V Atp5j V Atp5j2 V Atp5k V Atp5l VAtp5o V Atp5s V Atp5s1 V ENSMUSG00000064356 V ENSMUSG00000064357

TABLE 2 Genes included in Pathway Terms for FIGS. 1, 3A-3E, 3K, 4A, 4B,5A, 11-14, 20A, 20B Pathway Gene List Fatty acid Abcd2, Pex2, Cyp4f18,Slc27a2, Acot5, Slc27a5, Acox1, Acaa1a, metabolism Acot2, Acot4,Slc27a4, Elovl2, Acsbg1, AcotS, Pex5, Hsd17b4, Pla2g4f, Edn1, Edn2, Mif,Ptgis, Cd74, Ptgds, Pnpla8, Ptges2, Tbxas1, Ptges3, Ptges, Ptgs1, Hpgds,Fam213b, Ptgs2, Phyh, Pex13, Hacl1, Hao1, Acsl5, Cpt1a, Slc27a6,Slc27a1, Slc27a3, Acot1, Acsl4, Acsbg2, Acsl6, Acsl1, Acsl3, Acsm5,Fa2h, Ppara, Ghr, Prkar2b, Acaa2, Pla2g15, Agpat6, Acsf2, Snca, Them4,Amacr, Cryl1, Acsf3, Alkbh7, Cyb5a, Stat5b, Lypla1, Acnat2, Acox2, Aacs,Ankrd23, Cpt2, Baat, Abhd5, Cyp4a10, Crot, Acnat1, Angptl3, Mecr, Acsm4,Lypla2, Acot11, C3, Gpam, Sgpl1, Tnxb, Th, Apoa2, Crat, Aasdh, Cyp4a12a,Echdc2, Acsm2, Acaa1b, Stat5a, Crem, Ucp3, Ndufs6, Them5, Acot12, Scd1,Scd4, Ch25h, Lipc, Prkag1, Fads3, Prkag3, Acaca, Brca1, Ndufab1, Mlycd,Mcat, Prkaa2, Hnf1a, Acsm1, Sc5d, H2-Ke6, Agmo, Hsd17b12, Lpl, Mgl1,Acsm3, Prkab2, Abcd3, Msmo1, Prkaa1, Fasn, Pla2g1b, Olah, Cbr4, Prkag2,Pccb, Acly, Nr1h3, Fads6, Acacb, Degs1, Prkab1, Abcd1, Hadhb, Slc25a17,Cpt1c, Eci1, Lep, Acox3, Bdh2, Cpt1b, Adipoq, Decr1, Ppard, Eci3, Echs1,Ech1, Acadm, Pex7, Hadha, Ehhadh, Hadh, Sesn2, Fads1, 4833423E24Rik,Fads2, Elovl5, Elovl3, Decr2, Lta4h, Ltc4s, Cyp4f13, Alox5, Ncf1, Hpgd,Pdpn, Ptgr1, Ptgr2, Tnfrsf1a, Pdpn, Lpin1, Lpin2, Eci2, Faah, Acot7,Lpin3, Dld, Lias, Lipt2, Cyp4v3, Adh7, Elovl7, Elovl4, Elovl1, Elovl6,Alox15, Sstr4, Cyp1b1, Cyp2j5, Cyp4f14, Cyp2c54, Cyp2d12, Cyp2d11,Cyp2d34, Cyp2d22, Cyp2d9, Cyp2d40, Cyp2d10, Mapk3, Cyp2j13, Aloxe3,Alox8, Cyp2j9, Cyp2j11, Alox12b, Alox12e, Cyp2j8, Cyp2ab1, Alox12,Cyp2j6, Cyp2j12, Daglb, Dagla, Cyp2c55, Cyp2c50, Rnpepl1, Prg3, Cotl1,Ggt5, Fcer1a, Syk, 2010111101Rik, Ggt1, Mgst2, Pla2g5, Rnpep, Alox5ap,Pla2g3, Cyp2c69, Cyp2g1, Cyp2c44, Cyp2b23, Cyp2f2, Cyp2c70, Cyp2c66,Cyp2a22, Cyp2b19, Cyp2a12, Cyp2b13, Cyp2e1, Cyp2c38, Cyp2b10, Cyp2c40,Cyp2c39, Cyp2t4, Cyp2b9, Cyp2c67, Cyp2c29, Cyp2c37, Cyp2c68, Cyp2s1,Cyp2c65, Gcdh, Hao2, Mapk14, Adipor2, Cygb, Pparg, Adipor1, Por, Acss1,Acss2, Ces1f, Ces1d, Tecr, Hacd3, Hacd2, Pecr, Hacd1, Hacd4, Acad10,Acads, Acad9, Acadvl, Etfdh, Acad8, Acad12, Ivd, Acoxl, Etfa, Acadl,Acadsb, Acad11, Cyp4a31, Cyp4a32, Lipe, Asah2, Myo5a, Plp1, Qk, Tyrp1,Thnsl2, Aldh5a1, Oxsm, Scd3, Scd2 Glucose Akt1, Ppp1r3b, Mtor, Ppp1r3e,Ppp1ca, Ppp1r3d, Ppp1r3f, metabolism Ppp1cb, Inpp5k, Gnmt, Nr3c1, Nln,Lep, Pdk2, Fbp1, Slc35b4, Acadm, Rora, Mlycd, Lcmt1, Igfbp3, Fam132a,Igfbp5, Adipoq, Pdk1, Sirt1, Ncoa2, C1qtnf1, Pdk3, Pdk4, Igfbp4, Tff3,Nkx1-1, Adipor1, Rorc, Irs2, Irs1, Mup4, Pmaip1, Rgn, Mup9, Akt2, Mup20,Mup12, Mup19, Mup16, Foxa2, Ranbp2, Park2, Cox11, Gckr, Midn, Bad,Dusp12, Pfkfb1, Nr1d1, Foxo1, Tcf7l2, Supt20, Kat2b, Gcg, Wdr5, Kat2a,Pgam1, Tigar, Enpp1, Grb10, Pask, Clk2, Sik1, Serpina12, C1qtnf3, Lepr,Il6, Gek, Smek1, Ppara, Arpp19, Stk11, Smek2, Ptpn2, Pth, C1qtnf2,Dyrk2, Insr, Igf2, Ins2, Ppp1r3g, Igf1, Epm2aip1, Adra1b, Pomc, Khk,Gcgr, Phlda2, Ogt, Sesn2, Trp53, Actn3 Oxidative Ndufb11, Atp6v1h,Cox6b1, Atp6v1b1, Atp6v0b, Atp4a, Atp4b, phosphorylation Atp5a1, Atp5b,Atp5c1, Atp5f1, Atp5g1, Atp5j, Atp5k, Atp6v1a, Atp6v1b2, Atp6v0d1,Atp6v1e1, Atp6v0e, Atp6v0a1, Atp6v0c, Cox17, Cox4i1, Cox5a, Cox5b,Cox6a1, Cox6a2, Cox6c, Cox7a1, Cox7a2, Cox7c, Cox8a, Cox8b, Atp6v0a4,Atp6, Atp8, Cox1, Cox2, Cox3, Cytb, Nd1, Nd2, Nd3, Nd4, Nd4l, Nd5, Nd6,Ndufa2, Ndufa4, Ndufs4, Ndufv1, Atp12a, Cox7a2l, Atp6v0a2, Uqcrq,Uqcrc1, Ndufs8, Cox15, Ndufs2, Ndufs1, Atp5g3, Ndufb6, Gm4943, Atp6v0d2,Tcirg1, Atp5l, Atp5o, Cox6b2, Atp6v1g3, Ndufs6, Ndufa4l2, Ndufa1,Atp6ap1, Atp5j2, Ndufs5, Atp5d, Ndufb5, Sdhc, Ndufa3, Ndufa9, Cox7b,Atp6v1f, Uqcr10, Ndufb9, Atp6v1g2, Atp6v1g1, Atp6v1c1, Ndufc1, Ndufa12,Ndufa7, Cyc1, Ndufb3, Uqcrh, Uqcr11, Uqcrfs1, Ndufb7, Sdhd, Sdha,Uqcrc2, Atp5e, Ndufa6, Ndufa13, Ndufb8, Ndufa10, Uqcrb, Sdhb, Ppa1,Atp5g2, Ndufb4, Ndufc2, Ndufb2, Ndufa5, Ndufb10, Ndufs3, Ndufa8,Atp6v1c2, Cox11, Ndufa11, Ndufab1, Cox10, Atp5h, Ndufv2, Atp6v1d, Ppa2,Atp6v1e2, Ndufs7, Cox8c, Atp6v0e2, Lhpp, Cox7b2, Ndufv3, Cox4i2,ENSMUSG00000064341, ENSMUSG00000064345; ENSMGSG00000064360,ENSMUSG00000064363, ENSMUSG00000065947, ENSMUSG00000064367,ENSMUSG00000064368, ENSMUSG00000064370, ENSMUSG00000064351,ENSMUSG00000064354, ENSMUSG00000064358, ENSMUSG00000064357,ENSMUSG00000064356 Antioxidant Gpx1, Gpx2, Gpx3, Gpx4, Gpx5, Gpx6, Gpx7,Gstk1, Gstp1, Ehd2, metabolism Prdx1, Prdx2, Prdx3, Prdx4, Prdx5, Prdx6,Apc, Cat, Ctsb, Duox1, Epx, Lpo, Mpo, Ptgs1, Ptgs2, Rag2, Serpinb1b,Tpo, Alb, Gsr, Sod1, Sod3, Srxn1, Txnrd1, Txnrd2, Txnrd3 ROS Sod1, Sod2,Sod3, Ccs, Cyba, Ncf1, Ncf2, Nos2, Nox1, Nox4, metabolism Noxa1, Noxo1,Recql4, Scd1, Ucp2, Aox1, Fmo2, Il19, Il22, Als2, Apoe, Cat, Ccl5, Ctsb,Duox1, Epx, Ercc2, Ercc6, Fth1, Gclc, Gclm, Gpx1, Gpx2, Gpx3, Gpx4,Gpx5, Gpx6, Gpx7, Gsr, Gss, Hmox1, Hspa1a, Idh1, Krt1, Mpo, Nqo1, Park7,Prdx1, Prdx2, Prdx6, Prnp, Psmb5, Sod1, Sqstm1, Tpo, Txn1, Txnip,Txnrd1, Txnrd2, Ucp3, Xpa DNA damage Brca2, Ddb2, Dclre1a, Ercc1, Ercc2,Fancc, Lig1, Nthl1, Ogg1, and Pcna, Pole, Rpa1, Trp53, Xpa, Xpc, Apex1,Fen1, Lig1, Mbd4, repair Mpg, Nthl1, Ogg1, Parp1, Parp2, Pcna, Pole,Trp53, Ung, Xrcc1, Wrn, Abl1, Exo1, Mlh1, Mlh3, Msh2, Msh3, Pcna, Pms2,Atm, Blm, Brca1, Brca2, Chek1, H2afx, Hus1, Lig1, Mdc1, Mlh1, Mre11a,Nbn, Prkdc, Rad50, Rad51, Rad52, Rpa1, Trp53bp1, Xrcc2, Xrcc6, Atrx,Brip1, Chek2, Fanca, Fancd2, Fancg, Gadd45a, Gadd45g, Mgmt, Polh, Poli,Pttg1, Rad1, Rad17, Rad18, Rad21, Rad51c, Rad51b, Rad9a, Rev1, Rnf8,Smc1a, Smc3, Sumo1, Topbp1, Xrcc3 Mitochondrial Aip, Bak1, Bcl2, Bcl211,Bnip3, Cpt1b, Cpt2, Dnajc19, Timm10b, transport Grpel1, Hsp90aa1, Hspd1,Immp2l, Mfn2, Mipep, Mtx2, Stard3, Trp53, Tspo, Ucp1, Ucp2, Ucp3

(E) Top Enriched Pathways Based on IPA Analysis

Table 3 lists the 10 most enriched pathways based on IPA analysis. FIG.1 of U.S. Ser. No. 62/366,211, filed on Jul. 25, 2016, provides theresults of our preliminary analysis of the top enriched pathways betweenD2 mice and D2-Gpnmb⁺ control (the disclosure of which is incorporatedby reference).

TABLE 3 Ten most enriched pathways (IPA analysis) Rank PathwayComparison -log p value 1 EIF2 signaling D2 Group 2 vs. D2- 47.6 Gpnmb⁺2 Oxidative phosphorylation D2 Group 2 vs. D2- 26.6 Gpnmb⁺ 3Mitochondrial dysfunction D2 Group 2 vs. D2- 23.5 Gpnmb⁺ 4 Regulation ofeIF4 and D2 Group 2 vs. D2- 19.7 p70S6K Gpnmb⁺ signaling 5 mTORsignaling D2 Group 2 vs. D2- 18.3 Gpnmb⁺ 6 Phototransduction pathway D2Group 2 vs. D2- 8.8 Gpnmb⁺ 7 Fcγ receptor-mediated D2 Group 2 vs. D2-6.3 phagocytosis in Gpnmb⁺ macrophages and monocytes 8 IL-8 signaling D2Group 2 vs. D2- 5.1 Gpnmb⁺ 9 GABA receptor signaling D2 Group 2 vs. D2-4.9 Gpnmb⁺ 10 Huntington’s disease signaling D2 Group 2 vs. D2- 4.7Gpnmb⁺ 1 EIF2 signaling D2 Group 3 vs. D2- 30.4 Gpnmb⁺ 2 Oxidativephosphorylation D2 Group 3 vs. D2- 21.4 Gpnmb⁺ 3 Mitochondrialdysfunction D2 Group 3 vs. D2- 17.4 Gpnmb⁺ 4 mTOR signaling D2 Group 3vs. D2- 13.5 Gpnmb⁺ 5 Phototransduction pathway D2 Group 3 vs. D2- 11.8Gpnmb⁺ 6 Regulation of eIF4 and D2 Group 3 vs. D2- 11.6 p70S6K Gpnmb⁺signaling 7 Glutamate receptor signaling D2 Group 3 vs. D2- 5.6 Gpnmb⁺ 8Protein kinase A signaling D2 Group 3 vs. D2- 5.5 Gpnmb⁺ 9 Synaptic longterm potentiation D2 Group 3 vs. D2- 5.5 Gpnmb⁺ 10 Cardiac β-adrenergicsignaling D2 Group 3 vs. D2- 5.3 Gpnmb⁺ 1 EIF2 signaling D2 Group 4 vs.D2- 24.9 Gpnmb⁺ 2 Mitochondrial dysfunction D2 Group 4 vs. D2- 23.7Gpnmb⁺ 3 Oxidative phosphorylation D2 Group 4 vs. D2- 20.9 Gpnmb⁺ 4Axonal guidance signaling D2 Group 4 vs. D2- 14.0 Gpnmb⁺ 5 Regulation ofeIF4 and D2 Group 4 vs. D2- 13.2 p70S6K Gpnmb⁺ signaling 6 mTORsignaling D2 Group 4 vs. D2- 12.3 Gpnmb⁺ 7 CREB signaling in neurons D2Group 4 vs. D2- 11.0 Gpnmb⁺ 8 Signaling by Rho family D2 Group 4 vs. D2-9.35 GTPases Gpnmb⁺ 9 Huntington’s disease signaling D2 Group 4 vs. D2-9.1 Gpnmb⁺ 10 Breast cancer regulation by D2 Group 4 vs. D2- 9.0Stathmin1 Gpnmb⁺

Pathway analysis identified enrichment of eIF2 and mTOR signalingtranscripts (FIGS. 3B and 3F), with eIF2 signaling being the mostenriched pathway in Group 2 (the first distinguishable stage fromcontrols).

There was significant up-regulation of mitochondrial fission genes (Dnm1and Fis1) (FIG. 3D) and significant changes to the mitochondrialunfolded protein response (UPRmt) (FIG. 3E), further indicatingmitochondrial dysfunction.

(F) Morphology Reveals Mitochondrial Abnormalities

In this study, we evaluated morphological alternation by performingelectron microscopy (EM) on the retina in these mice. EM currently givesus the greatest resolution available to study the morphology ofintracellular organelles (such as mitochondria). EM revealeddysfunctional mitochondria with reduced mitochondrial cristae volume inthe dendrites of D2 RGCs, but not in those of control RGCs (FIGS. 4A and4B). These mitochondrial EM findings coincide with synapse loss in9-month old D2 retinas, with early decreases in patternelectroretinogram amplitude (PERG, a sensitive measure of RGC activityin both human patients and animals) (FIG. 14), and an increase inretinal cytochrome c levels (FIG. 3G).

These data presented clearly demonstrate that mitochondrialperturbations are among the very first changes occurring within RGCs invivo in an inherited age-related glaucoma. The present finding isconsistent with the reported in vitro study where cultured cells, whensubjected to pressure, undergo mitochondrial abnormalities. The in vitrostudy, however, fails to establish (while the present findingestablishes) how early the mitochondrial abnormalities occur in glaucomain vivo.

In summary, the present RNA-seq study revealed transcriptionalprocessing and mitochondrial dysfunction in the RGCs of glaucoma-proneeyes with undetectable neurodegenerative phenotype. Mitochondrialdysfunction was confirmed through targeted metabolic assays and EM,showing abnormal mitochondria early in glaucoma along with an earlyenergy crisis.

Example 2 Metabolic Profiling

To determine whether mitochondrial dysfunction/energy crisis was presentin D2 mice (as compared to the control D2-Gpnmb⁺ mice), during the pre-and the earliest stages of disease (i.e., at 4 months (pre-glaucoma), at9 months of age when high IOP is present without any neurodegeneration,and at 12 months when neurodegeneration is present/severe in themajority of eyes), we performed metabolomic profiling of neural retinas.

We performed additional metabolic profiling of neural retinas from 4-,9-, and 12-month D2 and D2-Gpnmb⁺ eyes. Metabolic profiling wasperformed using targeted assays following the manufacturersrecommendations. The following metabolites were profiled: NAD+/NADH(i.e. total NAD, NAD(t)), GSH/GSSG (i.e. total glutathione,glutathione(t)), and pyruvate. There were significant age-relateddecreases in all profiled metabolites. These metabolic profile changesoccur prior to any detectable neurodegenerative phenotype and also occurin age-matched, no glaucoma, control D2-Gpnmb⁺ retinas (FIG. 5A-D).

As these are key molecules in cellular metabolism and protection fromcellular stress, these age-dependent declines are expected to sensitizeretinal neurons to disease related stresses and mitochondrialdysfunction.

Our data show that HIF-1α (a key metabolic regulator during perturbedredox states) was induced in the ganglion cell layer early in glaucoma(by RNA-seq and by immunostaining: FIGS. 6A and 6B). This furthersuggests that RGCs undergo perturbed metabolism (and subsequent excessgeneration of reactive oxygen species) early in glaucoma.

To study the link between elevated IOP, mitochondrial dysfunction,metabolite depletion (especially NAD) and cellular stress, we examinedthe roles of DNA damage and PARPs (poly-ADP-ribose polymerase) in earlyglaucoma. PARPs respond to DNA-damage and are major consumers of NADwithin cells.

The RNA-seq dataset suggests that RGCs go through a period ofmitochondrial stress and metabolite depletion, potentially movingtowards fatty acid metabolism (corresponding with an increase in lipiddeposits in the retina during early glaucoma, FIGS. 7A and 7B).

One consequence of fatty acid β-oxidation is the increased generation offree radicals/reactive oxygen species (ROS). Thus, we further assessedretinas for signs of ROS-induced DNA damage (by RNA-seq, FIG. 3F; andγ-H2AX immunostaining, 3 sensitive marker of dsDNA breaks). γ-H2AX⁺nuclei were found to be present throughout the ganglion cell layer of D2retinas but not controls, indicating that DNA damage increases veryearly in glaucoma (FIGS. 8A and 8B).

PARP activity was found to be induced in RGCs with age (FIGS. 9A and9B), providing a link between DNA damage, and increased metabolic stresswithin RGCs. Our finding is consistent with the hypothesis that PARPsare induced by DNA damage, and they are major NAD⁺ consuming enzymes,and inhibit glycolysis through PAR inhibition of hexokinase.

The D2 mouse model clearly support the paradigm that age is major riskfactor for glaucoma and a variety of other neurodegenerative diseases.Our metabolic profile data suggest how increasing age may increaseneuronal vulnerability to damage through depletion of essential neuronalmetabolites.

Altogether, data presented herein support a new model whereage-dependent declines of NAD and glutathione in the retina sensitizeRGCs to glaucoma and possibly other age-related diseases. RGCs areparticularly vulnerable to harmfully high IOP and glaucoma. Thus,age-dependent metabolite decline, as well as PARP activation withinRGCs, conspire to disrupt cellular metabolism and increasesusceptibility to ongoing IOP-induced stresses, leading to criticaldamage of RGCs.

In D2 mice, glaucomatous neurodegeneration is age related, chronic andasynchronous. During development of D2 glaucoma, RGCs experience anearly loss of connectivity, as well as metabolic, and molecular changes,all of which occur prior to gross soma loss, axon loss, and degenerationof the optic nerve. These early changes are expected to decrease thereliability of cellular metabolism and increase the probability ofcellular failure with age when RGCs are under ongoing IOP-inducedstress.

In summary, based on RNA-sequencing and metabolic assays, we discoveredthat neurons in the retina go through a metabolic crisis prior todegenerating. This suggests a key role for metabolic molecules that mayalter or supplement the way mitochondria function. Correcting thesedysfunctional processes is likely to have benefits beyond glaucoma andrelate to more general age-related changes.

The data present so far shows that declining NAD is central toage-related neuronal vulnerability to glaucoma.

Example 3 NAM Protects Neuronal Cell Loss in Axotomy Culture

In this series of study, we examined if increasing the NAD levels canprotect insulted eyes from neurodegenerative changes. Axotomy (i.e. thesevering of the axon) mimics the acute severe insults seen in someglaucoma, and is an important model to test these more severe insults.

The study is based on the hypothesis that by decreasing the probabilityof metabolic/energetic failure, it would render the RGCs more resilientto the external stresses.

A plethora of drugs with actions at the mitochondria were tested inaxotomy culture, in order to identify drug candidates that potentiallyantagonize the mitochondrial dysfunction/energy crisis observed in theD2 mice. The screen identified nicotinamide (NAM) as giving the mostrobust protection against nuclear shrinkage, a hallmark feature ofnuclear remodeling pre-apoptosis. FIGS. 22A and 22B.

The above in vitro finding is consistent that the in vivo results inExample 3.

Example 4 NAM Alleviates Glaucoma in D2 Mice

We conducted an in vivo experiment using a large cohort of D2 mice.

In this experiment, optic nerves were classified into three damagelevels: NO (no glaucoma), MOD (Moderate damage), and SEV (severedamage).

Experimental D2 mice were divided into the following groups and weregiven:

-   -   W=water (standard mouse water)    -   NAM or NAM^(Lo)=550 mg/k/d NAM in drinking water    -   Early=early start=pre-glaucoma=6-month of age (i.e.        prophylactic)    -   Late=late start=during glaucoma=9-month of age (when mice        already have high IOP, i.e. interventional, and more relative to        human glaucoma)

NAD levels were increased by administering nicotinamide (NAM; aprecursor of NAD) to D2 mice (See. FIGS. 5A, 11-14, 20A, and 20B). Micewere then assessed at 12-month for optic nerve damage, soma loss, visualfunction, and axonal transport.

NAM administration in drinking water (550 mg/kg/d, NAM^(Lo) preventedthe decline of NAD levels through to 12-month (a standard end stage forassessing neurodegeneration in this glaucoma model) (FIGS. 5A and 5E).

(A) NAM Protects Mice from All Detectable Signs of Glaucoma

Supporting the neuronal vulnerability hypothesis, NAM did not alter IOP(FIGS. 10 A-10D), but it robustly protected from glaucoma, i.e. NAM is aneuroprotective agent. Importantly, NAM was protective bothprophylactically (starting at 6-month, early start; prior to IOPelevation in the vast majority of eyes in the colony), and as anintervention (starting at 9-month, late start; when the majority of eyeshave had continuing IOP elevation). This signifies that NAM is able toboth prevent neuronal injury from occurring as well as limiting neuronalinjury to already insulted neurons (FIGS. 11A and 11B). This isimportant for human disease where treatment would only start once adisease has become symptomatic and thus detectable.

These data support that NAM can be used prophylactically to treat humanglaucoma. Either in subjects at risk of glaucoma, for example, due tofamily history, ocular trauma, known gene mutation, or when IOP is seento be higher but no glaucomatous damage is present.

(B) NAM Robustly Prevents Retinal Ganglion Cell Dysfunction andDegeneration

NAM significantly reduced the incidence of optic nerve degeneration(FIGS. 11A and 12A), prevented RGC soma loss and retinal thinning(detectable in the human clinic through commonplace methods—e.g. OCT,fundoscopy) (FIGS. 12A and 13A), restored anterograde axonal transport(as assessed by Ct-β tracing; and important early marker of axondysfunction and degeneration) (FIG. 12A), and visual function asassessed by pattern electroretinography (PERG) (FIGS. 14 and 15), PERGhas been shown to be a very sensitive and early measure of glaucomatousvisual dysfunction in both humans and animal models (Saleh et al.,Invest Ophthalmol Vis Sci 48, 4564-4572, 2007); thus NAM prevents theearliest signs of glaucoma.

(C) NAM Protects Early Synapse Loss And Lipid Droplet Formation InRetina

NAM administration also protected against early synapse loss (SNAP-25immunostaining) that occurs in this model (FIGS. 16A and 16B), and wassufficient to inhibit the formation of dysfunctional mitochondria withabnormal cristae as assessed by EM (FIGS. 17A and 17B).

Lipid droplet formation was also prevented in aged D2 retinas (FIG. 18).

(D) NAM Decreases P RP Activation And Prevents Molecular Signs ofGlaucoma

NAM also decreased PARP activation, limited levels of DNA damage andtranscriptional induction of HIF-1α (FIGS. 9A, 9B, 19A and 19B)reflecting less perturbed cellular metabolism. NAM likely prevents thesedamaging mechanisms by correcting the upstream effectors—i.e. metabolitedepletion and redox buffering within the mitochondrion.

NAM prevented even the earliest molecular signs of glaucoma in themajority of treated eyes as assessed by RNA-seq. NAM treated sampleswere molecularly similar to controls and clustered among bothage-matched and young control samples (FIGS. 20A, 20B, and 21A-21F).

(E) NAM Prevents Age-Related Gene Expression In RGCs

NAM even prevented the vast majority of age-related gene expressionchanges within RGCs=number of DE genes; 4-month D2-GpNMB⁺ vs. 9-monthD2-Gpnmb⁺=4699, 9-month D2-GpnMB⁺ vs. NAM=4437, 4-month D2Gpnmb⁺ vs.NAM=83). This remarkable degree of molecular protection highlights theunexpected potency of NAM in decreasing the probability of metabolicdisruption and glaucoma in eyes with high IOP. As age is a major riskfactor in the pathogenesis of glaucoma, inhibiting damaging age-relatedchanges has the potential to prevent neurodegeneration in many instancesof human glaucoma as well as other slowly degenerating neurodegenerativediseases.

In the majority of the treated eyes, NAM administration completelyprevents glaucoma, including results based on very sensitive measures ofearly disease such as PERG. In addition, many ocular andneurodegenerative diseases occur in the elderly due to age-dependentmolecular chances that increase susceptibility to damage. NAM treatmentprevents age-related molecular changes assessed by gene expression (avery sensitive measure of these changes).

Furthermore, axonal degeneration and somal shrinkage may representcommon components in some neurodegenerative diseases. NAM prevents thesechanges based on the data above at least in glaucoma treatment.Furthermore, the data above shows that NAM prevents axonal degenerationand somal shrinkage in glaucoma.

Example 5 Increasing Dietary NAM Farther Lessens the Degree of IOPElevation in D2 Mice

NAM is believed to be safely tolerated even at high doses. Some studieshave suggested an incidence of hepatotoxicity at doses >4 grams NAM perday in humans. However, these resulted from impurities in olderpreparations. In a more recent survey of 6000 patients on high doses ofnicotinamide or niacin, only 3 cases of jaundice were reported. In oneof these (on 6 g/day niacin) it resolved after stopping a different butsimultaneously administered drug (but not niacin), and in another itresolved even with continuing niacin treatment. Regarding a higher doseof nicotinamide, it has been suggested that abnormal liver enzyme testsdo not indicate hepatocellular damage but rather represent changes inliver enzymes expression, which are rapidly reversible when the drug isdiscontinued. Rare cases of liver toxicity may reflect individualgenetic susceptibility or other individual factors. Although thelong-term effects of very high doses require further evaluation,experience suggests that risk to benefit ratios of long-termnicotinamide treatment would be highly favorable.

In an attempt to further decrease the probability of glaucoma andprotect even more eyes from IOP-induced insults, we used an increaseddose of NAM (representing 4 times the original lower dose at 2000mg/kg/d, NAM^(Hi)) administered to D2 mice.

Remarkably, NAM tat this dose) was found to be extremely protective with93% of treated eyes having no glaucomatous optic nerve damage (FIG.11A). This represents a ˜10 fold decrease in risk factor of developingglaucoma. The degree of protection afforded by administering this singlemolecule is unprecedented and completely unanticipated.

NAM at 550 mg/kg/d in mice demonstrates a clear neuroprotective effect(as IOP was not altered), the increased dose of 2000 mg/kg/d lessens thedegree of IOP elevation (FIGS. 10A and 10B). This indicates that NAMprotects against age-related pathogenic processes in additional celltypes to RGCs.

The present data indicate that NAM protects against both IOP elevationand neural vulnerability, and therefore has dual benefits and greatclinical potential in the treatment of human glaucoma. Even at the lower550 mg/kg/d dose in which IOP is not altered, IOP lowering treatments(such as surgery or eye-drops) could be combined with NAMsupplementation to provide greater protection against neurodegeneration.

Example 6 NAM is Effective in Two Models of RGC Death for GlaucomatousInsults

Glaucoma is a complex disease involving multiple insults. It has broadetiologies in which RGC compartments (e.g., soma, axon, dendrite) may bedifferently affected. Mechanical axon damage and local inflammationrepresent two important contributors to RGC degeneration duringglaucoma.

To assess the effectiveness of NAM treatment in different contexts, wetested NAM efficacy in two models of RGC death. The first glaucomatousinsult model involves the use of a tissue culture model of axotomy, andthe second glaucomatous insult model relates to the use of intravitrealinjections of soluble murine TNFα which drives local inflammation and isimplicated in glaucoma.

NAM robustly protected cultured retinas from RGC somal degeneration(FIGS. 22A and 22B), NAM also protected against a loss of PERG amplitudeand cell loss in TNFα injected eyes (FIGS. 23A-23C).

Given these protections against severe acute insults, and commonalitiesbetween glaucoma and other neurodegenerative diseases, NAM could havebroad implications for treating glaucoma and other age-relatedneurodegenerative diseases.

Example 7 Nmnat1Gene Therapy , Alleviates Glaucoma in D2 Mice

In this study, we demonstrate that overexpression of Nmnat1, a keyenzyme in NAD⁺ production (FIG. 25), supports NAD⁺ producing cellularmachinery.

We injected D2 mice eyes once at 5.5-month with AAV2.2 vector containingthe Nmnat1 gene and the GFP reporter, under a CMV promoter expressed asa single transcript. Mice were anaesthetised, and injectedintravitreally (i.e. behind the limbus, at a 45-60 degree angle into thevitreal chamber, avoiding the lens and central retina) with 1.5 μL ofviral vector (3×10⁸ U/mL). Both eyes were injected. Immediatelyfollowing injection hydrating eye drops were topically administered andmice were allowed to wake under a heat lamp. Eyes were clinicallyexamined at multiple time points following injection to confirm absenceof damage to the eye, Visual function assessment (by PERG) was performedprior to, and following, infection, confirming no adverse effects of theinitial injection and viral transfection.

Nmnat1 expression (as assessed by GFP expression) was detectable atleast as of the first week after AAV2.2 injection, and was robust inRGCs 2 weeks after injection (expressed in >83% of RGCs), and remainedrobust through to the end stage time point (12-month). The vast majorityof RGCs were transduced and expressed virally delivered gene products asevidenced by GFP fluorescence in RGC cell somas in the retina and theirterminal points in the brain, including the lateral geniculate nucleus(LGN) and superior collicuis (sup. col).

Overexpression of Nmnat1 was sufficient to prevent axon and soma loss(FIGS. 24A-24C, and FIG. 24D, upper panel) and to preserve axoplasmictransport and electrical activity in RGCs (PERG) (FIG. 24B and FIG.24D—lower panel). Glaucomatous nerve damage was absent in >70% oftreated eyes. This potent protection encourages the use of similar genetherapy strategies to prevent human glaucoma.

Example 8 Combination Therapy Using Nmnat1 Gene Therapy and NAM

In this study, we examined the effects of combinational therapy ofNmnat1 and NAM (NAM^(Lo)) by combining the experiments detailed inExample 4 and Example 7, Briefly, mice underwent gene therapy as above,following a 1 week viral shedding period mice were returned to our usualcolony and administered NAM 550 mg/kg/d in normal drinking water.

This combination afforded significant additional protection with 84% ofeyes having no detectable glaucomatous damage at 12 months of age. Thisrepresents a ˜4-fold decrease in the risk of developing IOP inducedglaucomatous neurodegeneration compared to untreated D2 controls.

Increasing doses of NAM in combination with gene therapy is even moreprotective.

Example 9 Wld^(S) Alleviates Glaucoma in D2 Mice

The Wallerian degeneration slow allele (Wld^(S) ) has been show topartially protect in D2 glaucoma. The WLD^(s) protein is a modifiedNMNAT1 protein (an enzyme which converts NMS to NAD). Cells containingthe mutant protein WLD^(s) show increased enzymatic activity, convertingNMN to NAD either quicker or more efficiently.

In this experiment, we demonstrated that NAM and Wld^(S) combinationbetter protects against glaucoma than Wld^(S) alone, in D2 mice bearingthe Wld^(S) mutation. Mice administered NAM and carrying the Wld^(s)mutation show profound protection from glaucomatous damage (˜95% noglaucoma).

The experiment was run essentially the same as in Example 4, except thatD2 mice with the Wallerian degeneration slow allele (Wld^(S) ) wereused. The results were also shown in FIG. 11B. It is evident that eitherWld^(S) alone or NAM alone protects against D2 glaucoma, but thecombination of Wld^(S) and NAM is significantly better than Wld^(S)alone.

Specifically, NAM alone is about 70% protective in glaucoma. Meanwhile,NAM+Wld^(S) is ˜95% protective in glaucoma. Both NAM and NAM+Wld^(S)protect all portions of the cell and optic nerve (data not shown). Thus,while NAM and Wld^(S) are partially protective individually, thecombination exhibited a significant synergistic effect (i.e., from ˜70%protection to ˜95%).

While not wishing to be bound by any particular theory, it is believedat increasing Nmnat expression by using the Wld^(S) mutation allows moreNAM to be converted to NADt, and the combination protects againstneurodegeneration in glaucoma synergistically.

Consistent with this theory, in DBA/2J mice, there is an age and diseaserelated decrease in cellular levels of NADt and other TCA/Krebs Cyclecomponents (such as pyruvate, FIG. 5D). Thus the cellular levels of NADt(NAD⁺/NADH) was determined in treated animals.

We found that levels of NADt (NAD⁺/NADH) were restored in NAM treatedmice as well as in Wld^(S) mice. When Wld^(s) mice is further treatedwith NAM, the restoration of NADt level is synergistic in NAM+Wld^(S)treated mice. (See FIG. 5E).

The result of this example is consistent with that in Example 8.

NADt (NAD⁺/NADH) levels are thought to change in aging andneurodegenerative diseases. The data provided herein demonstrates thatadministration/supplementation of NAM, NAM derivatives, and/or NMNATenzyme may be protective in a large range of aging and diseasephenotypes.

Example 10 Pyruvate Alleviates/Prevents Glaucoma in D2 Mice

This example demonstrates that nicotinamide (Vitamin B3) and/or incombination with pyruvate (a simple metabolite of glucose) robustlyprotect DBA/2J mice from vision loss, loss of axonal transport, retinaand optic nerve damage. Gene expression experiments demonstrate thatmice treated with nicotinamide are more closely molecularly matched toyoung control mice, rather than age-matched non-glaucomatous mice. Thedata suggests that nicotinamide may be working in part through anage-dependent mechanism, delaying age dependent increases insusceptibility to glaucoma. The magnitude of protection that NAMprovides is completely unexpected and surprising.

Pyruvate is an important simple alpha-keto acid involved in metabolismwhere it is produced from glucose. Pyruvate is converted intoacetyl-coenzyme A, the main input for the Kreb's cycle (citric acidcycle). During normoxia (normal conditions) pyruvate increases NADHlevels.

Pyruvate levels decrease in the retina with age, sensitizing retinalneurons to glaucomatous damage from high intraocular pressure (FIG. 5D).Mice administered pyruvate in normal drinking water had increased levelsof pyruvate in the retina (FIG. 5D). Mice administered pyruvate innormal drinking water were resistant to optic nerve axon degeneration,cell loss and visual dysfunction (as assessed by PERG) in glaucoma(FIGS. 11B, 12B, 13B, and 14).

The findings above showed increased potency in mice both administeredNAM and pyruvate, demonstrating a synergistic effect between NAM- andpyruvate-treatment to prevent neuronal degeneration.

Example 11 PQQ Prevents Nuclear Diameter Shrinkage and Decrease in CellDensity

Additional neuroprotective agents were also tested to determine theirroles in neurodegenerative diseases.

In one experiment, similar experiments were set up to determine theneuroprotective function of pyrroloquinoline quinone (PQQ). PQQ is animportant redox cofactor, similar to nicotinamide. PQQ promotesmitochondrial biogenesis, and its neuroprotective function is thought todepend on its functions as an antioxidant. FIG. 26 shows that PQQ isprotective in retina tissue culture.

Example 12 Glutathione Levels in the Retina Decrease with Age

N-acetylcysteine (N-acetyl-L-cysteine or NAC) is a precursor toL-cysteine, a precursor to glutathione, a potent biological antioxidant,and a major redox buffer in neurons. Data in FIG. 5B shows thatglutathione levels in the retina decrease with age.

NADPH (produced from NAD) is required for GSH synthesis from GSSG. Thus,NAD and GSH levels are intrinsically linked.

MATERIALS AND METHODS 1. Mouse Strains, Breeding and Husbandry

Mice were housed in a 14-hr light/10-hr dark cycle with food and wateravailable ad libitum as previously reported (Howell et al., Journal ofClinical Investigation 121, 1429-1444, 2011). All breeding andexperimental procedures were undertaken in accordance with theAssociation for research for Vision and Ophthalmology Statement for theUse of Animals in Ophthalmic Research. The Institutional BiosafetyCommittee and the Animal Care and Use Committee at The JacksonLaboratory approved this study.

C57BL/6J (B6), DBA/2J (D2), and DBA/2j-Gpnmb^(R150X) (D2-Gpnmb⁺) strainswere utilized and have been described in detail elsewhere (Anderson etal., Nature Genetics 30, 81-85, 2002). D2-Gpnmb⁺ mice do not develophigh IOP and thus do not develop glaucomatous neurodegeneration (Libbyet al., Vis Neurosci 22, 637-648, 2005). They are a genetically matchedcontrol for DBA/2J mice.

For aged glaucoma experiments mice were administered NAM in food and/orwater starting at 6-month (prophylactic, prior to IOP elevation inalmost all eyes) or 9-month (when the majority of eyes have high IOP andmolecular changes, but no detectable neurodegeneration) (Howell et al.,Journal of/Clinical Investigation 121, 1429-1444, 2011). These molecularchanges are prior to PERG defects and loss of anterograde axoplasmictransport. Low dose NAM (NAM^(Lo), 550 mg/kg/d, PanReac AppliChem) wasdissolved in regular acid drinking water (350 mL) and changed once perweek. For high-dose NAM (NAM^(Hi), 2000 mg/kg/d), NAM was available inboth water (550 mg/kg/d) and food (1450 mg/kg/d) and was changed onceper week. NAM food was prepared in a previously published, customWestern diet for palatability (LabDiet) (Graham et al., Sci Rep 6,21568, 2016). Control mice received the same diet with no added NAM.This diet has no effects on the ocular health of the mice.

2. FAC sorting of RGCs

Prior to cell collection, all surfaces and volumes were cleaned with 70%ethanol and RNaseZap (ThermoFisher Scientific) solution followed bydH₂O. Mice were euthanized, eyes enucleated and placed immediately intoice-cold HBSS. Retinas were dissected from the eyes (4- or 9-month ofage, no axon degeneration confirmed by PPD staining, data not shown) inHBSS on ice and placed directly into 100 μL, of a custom HBSS (Gibco),dispase (5 U/mL) (Stemcell Technologies), DNase 1 (2000 U/mL)(Worthington Biochemical) and SUPERase (1 U/μL) (ThermoFisherScientific) solution. All retinas were from eyes that had noglaucomatous axon degeneration by PPD staining and analysis (see opticnerve assessment below). Retinas were incubated for 20 mins at 37° C.and shaken at 350 RPM in an Eppendorf Thermomixer R followed by gentletrituration using a 200 μL pipette. Samples were blocked in 2% BSA,SUPERase (1 U/μL) in HBSS, and stained with conjugated antibodiesagainst Cd11b, Cd11c, Cd34, Cd45.2, GFAP, Thy1.2 as well as DAPI. Thiscocktail allowed other retina cell types to be accurately removed duringFACS.

FACS was performed on a FACSAria (BD Biosciences). Thy1.2⁺ (and negativefor all other markers) RGCs were sorted into 300 μL buffer RLT+1% β-ME,vortexed and frozen at −80° C. until further processing.

3. RNA-Sequencing

RAC sorted RGC samples were defrosted on ice and homogenized by syringein RLT Buffer (total volume 300 μL). Total RNA was isolated using RNeasymicro kits according to manufacturer's protocols (Qiagen) including theoptional DNase treatment step, and quality was assessed using an Agilent2100 Bioanalyzer. The concentration was determined using Ribogreen Assay(Invitrogen). Amplified dscDNA libraries were created using a NugenOvation RNA-seq System V. The SPIA dscDNA was sheared to 300 bp inlength using a Diogenode Disruptor. Quality control was performed usingan Agilent 2100 Bioanalyzer and a DNA 1000 chip assay.

Library size produced was analyzed using qPCR using the LibraryQuantitation kit/Illumina GA/ABI Prism (Kapa Biosystems). Libraries werebarcoded, pooled, and sequenced 6 samples per lane on a HiSeq 2500sequencer (illumina) giving a depth of 30-35 million reads per sample.

4. Differential Gene Expression and Pathway Analysis

Samples were subjected to quality control analysis by a custom qualitycontrol python script. Reads with 70% of their bases having a basequality score ≥30 were retained for further analysis. Read alignment wasperformed using TopHat v 2.0.7 (Kim et al., Genome Biol 14, R36, 2013)and expression estimation was performed using HTSeq (Anders et al.,Bioinformatics 31, 166-169, 2015) with supplied annotations and defaultparameters against the DBA/2J mouse genome (build-mm10). Bamtools v1.0.2 (Barnett el al., Bioinformatics 27, 1691-1692, 2011) were used tocalculate the mapping statistics.

Differential gene expression analysis between groups was performed usingedgeR v 3,10.5 (Robinson et al., Bioinformatics 26, 139-140, 2010)following, batch correction using RUVSeq, the removal of outlier samplesand lowly expressed genes by removing genes with less than five reads inmore than two samples. Normalization was performed using the trimmedmean of M values (TMM).

Unsupervised HC was performed in R (1-cor, Spearman's rho). A total of72 samples across all groups successfully amplified and sequenced andfollowing HC pipelines 9 samples were removed as outliers. Adjustmentfor multiple testing was performed using false discovery rate (FDR).Genes were considered to he significantly differentially expression atan FDR <0.05.

To study age-related changes, comparisons were made between 4-month and9-month D2-Gpnmb⁺ samples and NAM^(Lo) samples. For pathway analysis,QIAGEN's Ingenuity Pathway Analysis (IPA, Qiagen) was used for networkgeneration across genes that are significantly differentially expressed.Lists of significantly differentially expressed genes were uploaded toIPA and mapped to the Mus musculus Entrez gene symbols using the IPAknowledge base. These objects will then be overlaid onto a canonicalpathways developed from information contained in the IPA Knowledge Base.IPA's inbuilt network scoring algorithm will be used to rank thenetworks generated.

5. Metabolic Phenotyping

For GSH/GSSO and NAD⁺/NADH quantification retinas were dissociated (asabove) and compounds measured following the manufacturer's instructions(GSH/GSSG, Cayman Chemical; NAD⁺/NADH, Biovision). Results werecalculated according to the standard curve generated by using standardsfrom the kits. Final metabolite concentrations for each samples werenormalized to total protein concentration measured by Bradford assay.

6. Whole Retina Explant Culture

Mice were euthanized, eyes enucleated and placed immediately intoice-cold HBSS. Retinas were dissected from the eyes in HBSS on ice,flat-mounted with the ganglion cell layer up on a cell culture insert(Millipore), and submerged in tissue culture media containingNeurobasal-A, 1% penicillin-streptomycin (10000 U/mL), 1% glutamine(100×), 1% N-2 (100×) and 1% B-27 (50×) (all ThermoFisher Scientific).

For drug treated retinas tissue culture media was made as above andsupplemented with one of the following compounds (all Sigma unlessotherwise): β-NAD, NAM (PanReac AppliChem), β-NMN. Retinas wereincubated in 6-well plates at 37° C. and 4% CO₂ for 5 days before beingfixed in 4% PFA and stained with DAPI. (For untreated “Day 0” controls,retinas were dissected and placed straight into 4% PFA.) Retinas wereimaged on a Zeiss AxioObserver.

7. Soluble Murine TNF α Injections

To induce delayed retinal ganglion cell degeneration, soluble murineTNFα (1 ng/μL) was intravitrally injected as previously described(Nakazawa et al., J Neurosci 26, 12633-12641, 2006). Ten week old B6mice were pre-treated with NAM for 2 weeks prior to TNFα injections andPERG was performed at 8, 10 and 12 weeks of age. Mice we sacrificed at12-week and RGC counts performed.

8. Clinical Phenotyping

IOP elevation in D2 mice is subsequent to a pigment dispersing irisdisease. In all experiments, the progression of the iris disease andintraocular pressure in mutant or drug-treated mice was compared tocontrol D2 mice as previously described (John et al., Invest OphthalmolVis Sci 39, 951-962, 1998). In each experiment, iris disease andintraocular pressure were assessed.

Iris disease was assessed at two-month intervals starting at 6 months ofage until experiment completion.

Intraocular pressure was measured at 45-day intervals beginning at 8.5-9month until experiment completion.

9. Pattern Electroretinography (PERG)

PERG was recorded subcutaneously from the snout as previously reported.Briefly: patterned stimuli (gratings of 0.05 cycles/degree, 100%contrast) generated on LED panels were presented at each eye separatelywith slight different frequencies around 1 Hz. Waveforms were retrievedusing an asynchronous averaging method (Chou et al., Invest OphthalmolVis Sci 55, 2469-2475, 2014).

Mice were anaesthetized using ketamine/xylazine (Savinova et al., BMCGenet 2, 12, 2001) and body temperature maintained at 37° C. on afeedback-controlled heated stage monitored by rectal thermometer.

10. Optic Nerve Assessment and Determination of Glaucomatous Damage

The processing of optic nerves and staining with paraphenylenediamine(PPD) was as published (Smith et al., Systematic evaluation of the mouseeye. Anatomy, pathology and biomethods. CRC Press, Boca Raton, 2002).PPD stains the myelin sheath of all axons but darkly stains the axoplasmof only damaged axons. It is well established to provide a verysensitive measure of optic nerve damage (Smith et al., Systematicevaluation of the mouse eye. Anatomy, pathology and biomethods. CRCPress, Boca Raton, 2002).

Briefly, intracranial portions of optic nerves were fixed in 4% PFA atRT for 48 hrs, processed and embedded in plastic. A segment of opticnerve from within a region up to 1 aim from the posterior surface of thesclera was sectioned (1 μm thick sections) and stained with PPD.Typically 30-50 sections are taken from each nerve.

Multiple sections of each nerve were considered when determining damagelevel. Optic nerves were analyzed and determined to have one of 3 damagelevels:

(1) No or early damage (NOE) less than 5% axons damaged and no gliosis.This level of damage is seen in age and sex matched non-glaucomatousmice and is not due to glaucoma. Although none of these eyes exhibitglaucomatous nerve damage, this damage level is called no or earlyglaucoma as some of these eyes have early molecular changes that precedeneurodegeneration. These molecular changes can be detected by geneexpression studies (Howel (el al., Journal of Clinical Investigation121, 1429-1444, 2011). Eyes with these early molecular changes but nodegeneration are considered to have early glaucoma when discussingmetabolic, mitochondrial and gene expression changes herein.

(2) Moderate damage (MOD)—average of 30% axon loss and early gliosis.

(3) Severe (SEV)→50% axonal loss and damage with prominent gliosis.

11. Anterograde Axon Transport

Mice were anaesthetized using ketamine/xylazine and intravitreallyinjected with 2 μL AF488 or AF594 cholera toxin subunit B (1 mg/mL inPBS) (ThermoFisher Scientific). After 72 hrs mice were anaesthetized andeuthanized via 4% PFA cardiac perfusion. Brains and eyes were post-fixedin 4% PFA fir an additional 24 hrs, cryoprotected in 30% sucrose in PBSovernight (ON), OCT cryoembedded and sectioned at 20 μm. AF488 wasvisualized using a Zeiss AxioObserver or Zeiss AxioImager.

12. Histology

For immunofluorescence staining, mice were euthanized by cervicaldislocation, their eyes enucleated and placed in 4% PFA ON. Retinas weredissected and flat-mounted onto slides, permeabilized with 0.1% Triton-Xfor 15 mins, blocked with 2% BSA in PBS and stained ON at RT in primaryantibody (see below for list of antibodies).

After primary antibody incubation, retinas were washed 5 times in PBS,stained for 4 hrs at RT with secondary antibody. Slides were then washeda further 5 times with PBS, stained with DAPI for 15 mins, mounted withfluoromount, coverslipped and sealed with nail-polish.

For retinal sections, eyes were cryoprotected in 30% sucrose ON, frozenin OCT and cryosectioned at 18 μm. Slides were warmed to roomtemperature and the procedure above was followed. Retinas were imaged ona Zeiss AxioObserver for low resolution counts. Retinal sections wereimaged on a Leica SP8 far higher resolution images.

For Nissl staining, frozen sections were warmed to room temperature,placed in 1:1 alcohol:chloroform ON, and rehydrated through serialalcohol gradient. Slides were washed once in distilled water and stainedfor 15 mins in 0.1% cresyl violet in distilled water before beingdifferentiated in 95% alcohol, dehydrated in 100% alcohol and cleared inxylene. Slides were prepared as above. Nissl stained retinal sectionswere imaged using a Nikon Eclipse E200.

For Oil Red O staining, slides were warmed to room temperature, washedquickly in 60% isopropanol, incubated at RT in Oil Red O andHaematoxylin for 15 mins, washed in 60% isopropanol again, mounted andcoverslipped. Oil Red O stained retinal sections were imaged using aNikon Eclipse E200.

13. Western Blotting

Mice were euthanized by cervical dislocation, eyes enucleated and placedimmediately into ice-cold HBSS. Retinas were dissected from the eyes inHBSS, placed directly in to RIPA buffer and homogenized. Proteinextracts separated by SDS-PAGE in precast gels (Bio-Rad) and transferredonto PVDF membranes using an iBlot 2 (ThermoFisher Scientific).Membranes were blocked in 5% milk or 5% BSA in 0.1% PBS-Tween andantibody incubations were performed in blocking solution. Antibodystaining was detected using ECL. Protein concentration was assessed byBradford assay and normalized to a housekeeping protein usingdensitometry.

14. Electron Microscopy

Mice were euthanized, eyes enucleated, and then fixed ON in Smith-Ruda(0.8% PFA and 1.2% gluteraldehyde in 0.1 M phosphate buffer). The corneaand lens were dissected off and the remaining posterior eye-cup(containing the retina and ONH) were post-fixed in 2% aqueous osmiumtetroxide and dehydrated in ethyl alcohol. Samples were then infiltratedand flat-mounted in Embed 812/Araldite resin (Electron MicroscopySciences) to allow en face sections. Blocks were polymerized at 60° C.for 48 hrs and 90 nm microtome sections were cut (Leica UC6, Leica) onto300 mesh copper grids. Grids were stained with 1% aqueous uranylacetate/Reynold's lead citrate and were viewed on a JEOL JEM 1230 TEM.Images were collected on an AMT 2K camera.

15. Gene Therapy

For virally delivered gene therapy, mice were anaesthetized (as above),and intravitreally injected with 1.5 μL (3.1×10¹⁰ gc/mL) AAV2.2 murineNmnat1 under a CMV promoter with a GFP reporter (referred to as Nmnat1in main text; Vector Biolabs #ADV-265880). Mice were injected at5.5-month of age in a BSL2 laboratory and moved to our regular mousecolony after 2 weeks.

For Nmnat1 mice undergoing additional NAM treatment, mice were given 550mg/kg/d NAM in normal drinking water from 6-month of age. Mice wereeuthanized at 12-month.

16. Antibodies

Antibodies used in the examples above are listed below. IF:immunofluorescent. WB: Western Blot

Antibody Use Concentration Manufacturer Catalogue # CYCS WB 1:1000 Abcamab90529 EIF2 WB 1:1000 Cell Signaling 97225 H2AX IF 1:500 Millipore05-636 HIF1A IF 1:500 Novus Biologicals NB100-105 PARP IF 1:500 BDPharmingen 556362 RBPMS IF 1:500 Novus Biologicals NBP2-20112 SNAP-25 IF1:250 Abcam ab24737

17. Statistical Analysis

The sample size (number of eyes, n) is shown in each figure legend.Graphing and statistical analysis was performed in R. Student's t testwas used for pairwise analysis in quantitative plots and error barsrefer to standard error of the mean unless otherwise stated.

All cited references are incorporated by reference. While specificembodiments of the present invention have been described in theforegoing, it will be appreciated by those skilled in the art that manyequivalents, modifications, substitutions, and variations may be madethereto without departing from the spirit and scope of the invention asdefined in the appended claims.

1. A method of reducing axonal degeneration of a retinal ganglion celland treating glaucoma in a subject in need thereof, comprising the stepof orally administering to the subject a therapeutically effectiveamount of nicotinamide (NAM) and pyruvate to reduce axonal degenerationin a retinal ganglion cell and treat the glaucoma, wherein the subjectis a mammal and administered between 1 and 5 g/day of NAM and between 1and 5 g/day of pyruvate.
 2. The method of claim 1, wherein the subjectis also administered one or more compounds selected from the groupconsisting of nicotinamide mononucleotide (NMN), pyrroloquinolinequinone (PQQ), nicotinamide adenine dinucleotide (NAD) and nicotinamideribose (NR).
 3. The method of claim 1, wherein the NAM is present in atherapeutically effective amount to reduce intraocular pressure.
 4. Themethod of claim 1, wherein the NAM and pyruvate are present intherapeutically effective amounts to reduce neurodegeneration in aretinal ganglion cell, or to reduce intraocular pressure.
 5. The methodof claim 1, wherein the subject is a human subject.
 6. The method ofclaim 1, the method further comprises the step of administering a genecomposition, wherein said gene composition comprises a polynucleotideencoding NMNAT1.
 7. The method of claim 6, wherein the polynucleotide isin a viral vector.
 8. The method of claim 7, wherein the viral vector isan adeno-associated virus (AAV) vector, an adenoviral vector, alentiviral vector, or a retroviral vector.
 9. The method of claim 7,wherein the viral vector is an AAV vector.
 10. The method of claim 7,wherein the viral vector is a lentiviral vector.
 11. The method of claim6, wherein the gene composition is administered intravitreally orintraocularly.
 12. The method of claim 6, wherein the gene compositionis administered intravitreally.
 13. The method of claim 1, the methodfurther comprising the step of administering a gene composition, whereinthe gene composition comprising a polynucleotide encoding NMNAT1. 14.The method of claim 1, further comprising administering to the subjectan additional therapeutic agent.
 15. The method of claim 14, wherein theadditional therapeutic agent is a beta blocker, a nonselectiveadrenergic agonist, a selective a-2 adrenergic agonist, a carbonicanhydrase inhibitor, a prostaglandin analog, a para-sympathomimeticagonist, a carbachol or a combination thereof.
 16. The method of claim14, wherein the additional therapeutic agent is timolol, levobunolol,metipranolol carteolol, betaxolol, epinepherine, apraclonidine,brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide,latanoprost, travaprost, bimataprost, pilocarpine, echothiophate iodide,carbachol, or a combination thereof.
 17. A method of preventing glaucomain a subject in need thereof, comprising the step of orallyadministering to the subject a pharmaceutical composition containing atherapeutically effective amount of nicotinamide (NAM) and pyruvate toreduce axonal degeneration in a retinal ganglion cell and preventglaucoma, wherein the subject is a mammal and is administered between 1and 5 g/day of NAM and between 1 and 5 g/day of pyruvate.
 18. A methodof improving visual function in a subject in need thereof, comprisingthe step of orally administering to the subject a pharmaceuticalcomposition containing a therapeutically effective amount ofnicotinamide (NAM) and pyruvate to reduce axonal degeneration in aretinal ganglion cell and improve visual function, wherein the subjectis a mammal and is administered between 1 and 5 g/day of NAM and between1 and 5 g/day of pyruvate.
 19. The method of claim 17, wherein thesubject is a human subject.
 20. The method of claim 18, wherein thesubject is a human subject.